Search results for the GEO ID: GSE7850  |
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GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM190495 | GPL201 |
|
Retinal endothelial cell_Human 1_Unstimulated_Replicate 1
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 4 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 1ug; Hybridization date: 10/08/2004; Array lot: 4003908
|
| Sample_geo_accession | GSM190495
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190495/suppl/GSM190495.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190496 | GPL201 |
|
Retinal endothelial cell_Human 1_Stimulated.Toxoplasma_Replicate 1
|
Retinal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 4 years
Cell: retinal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1ug; Hybridization date: 10/08/2004; Array lot: 4003908
|
| Sample_geo_accession | GSM190496
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190496/suppl/GSM190496.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190497 | GPL201 |
|
Retinal endothelial cell_Human 1_Stimulated.Toxoplasma_Replicate 2
|
Retinal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 4 years
Cell: retinal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1ug; Hybridization date: 10/08/2004; Array lot: 4003908
|
| Sample_geo_accession | GSM190497
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190497/suppl/GSM190497.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190498 | GPL201 |
|
Choroidal endothelial cell_Human 1_Unstimulated_Replicate 1
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 4 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 1ug; Hybridization date: 10/08/2004; Array lot: 4003908
|
| Sample_geo_accession | GSM190498
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190498/suppl/GSM190498.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190499 | GPL201 |
|
Choroidal endothelial cell_Human 1_Unstimulated_Replicate 2
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 4 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 1ug; Hybridization date: 10/08/2004; Array lot: 4003908
|
| Sample_geo_accession | GSM190499
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190499/suppl/GSM190499.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190500 | GPL201 |
|
Choroidal endothelial cell_Human 1_Stimulated.Toxoplasma_Replicate 1
|
Choroidal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 4 years
Cell: choroidal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1ug; Hybridization date: 10/08/2004; Array lot: 4003908
|
| Sample_geo_accession | GSM190500
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190500/suppl/GSM190500.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190501 | GPL201 |
|
Choroidal endothelial cell_Human 1_Stimulated.Toxoplasma_Replicate 2
|
Choroidal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 4 years
Cell: choroidal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1ug; Hybridization date: 10/08/2004; Array lot: 4003908
|
| Sample_geo_accession | GSM190501
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190501/suppl/GSM190501.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190502 | GPL201 |
|
Retinal endothelial cell_Human 2_Unstimulated_Replicate 1
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 37 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190502
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190502/suppl/GSM190502.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190503 | GPL201 |
|
Retinal endothelial cell_Human 2_Unstimulated_Replicate 2
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 37 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190503
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190503/suppl/GSM190503.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190504 | GPL201 |
|
Retinal endothelial cell_Human 2_Stimulated.Toxoplasma_Replicate 1
|
Retinal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: female
Age: 37 years
Cell: retinal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
|
|
GSM190505 | GPL201 |
|
Retinal endothelial cell_Human 2_Stimulated.Toxoplasma_Replicate 2
|
Retinal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: female
Age: 37 years
Cell: retinal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190505
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190505/suppl/GSM190505.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190506 | GPL201 |
|
Choroidal endothelial cell_Human 2_Unstimulated_Replicate 1
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 37 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190506
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190506/suppl/GSM190506.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190507 | GPL201 |
|
Choroidal endothelial cell_Human 2_Unstimulated_Replicate 2
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 37 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190507
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190507/suppl/GSM190507.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190508 | GPL201 |
|
Choroidal endothelial cell_Human 2_Stimulated.Toxoplasma_Replicate 1
|
Choroidal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: female
Age: 37 years
Cell: choroidal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190508
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190508/suppl/GSM190508.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190509 | GPL201 |
|
Choroidal endothelial cell_Human 2_Stimulated.Toxoplasma_Replicate 2
|
Choroidal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: female
Age: 37 years
Cell: choroidal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 1.5ug; Hybridization date: 03/01/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190509
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190509/suppl/GSM190509.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190510 | GPL201 |
|
Retinal endothelial cell_Human 3_Unstimulated_Replicate 1
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 44 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 06/17/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190510
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190510/suppl/GSM190510.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190511 | GPL201 |
|
Retinal endothelial cell_Human 3_Stimulated.Toxoplasma_Replicate 1
|
Retinal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 44 years
Cell: retinal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 2ug; Hybridization date: 06/17/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190511
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190511/suppl/GSM190511.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190512 | GPL201 |
|
Retinal endothelial cell_Human 3_Stimulated.Toxoplasma_Replicate 2
|
Retinal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 44 years
Cell: retinal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 2ug; Hybridization date: 06/17/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190512
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190512/suppl/GSM190512.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190513 | GPL201 |
|
Choroidal endothelial cell_Human 3_Unstimulated_Replicate 1
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 44 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 06/17/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190513
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190513/suppl/GSM190513.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190514 | GPL201 |
|
Choroidal endothelial cell_Human 3_Unstimulated_Replicate 2
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 44 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 06/17/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190514
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190514/suppl/GSM190514.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190515 | GPL201 |
|
Choroidal endothelial cell_Human 3_Stimulated.Toxoplasma_Replicate 1
|
Choroidal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 44 years
Cell: choroidal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 2ug; Hybridization date: 06/17/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190515
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190515/suppl/GSM190515.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190516 | GPL201 |
|
Choroidal endothelial cell_Human 3_Stimulated.Toxoplasma_Replicate 2
|
Choroidal endothelial cell, Toxoplasma gondii, 4 hours
|
Gender: male
Age: 44 years
Cell: choroidal endothelial
Stimulus: Toxoplasma gondii
|
RNA input amount: 2ug; Hybridization date: 06/17/2005; Array lot: 4005095
|
| Sample_geo_accession | GSM190516
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190516/suppl/GSM190516.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190517 | GPL201 |
|
Retinal endothelial cell_Human 4_Unstimulated_Replicate 1
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 36 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190517
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190517/suppl/GSM190517.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190518 | GPL201 |
|
Retinal endothelial cell_Human 4_Unstimulated_Replicate 2
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 36 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190518
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190518/suppl/GSM190518.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190519 | GPL201 |
|
Retinal endothelial cell_Human 4_Stimulated.LPS_Replicate 1
|
Retinal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 36 years
Cell: retinal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190519
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190519/suppl/GSM190519.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190520 | GPL201 |
|
Retinal endothelial cell_Human 4_Stimulated.LPS_Replicate 2
|
Retinal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 36 years
Cell: retinal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190520
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190520/suppl/GSM190520.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190521 | GPL201 |
|
Choroidal endothelial cell_Human 4_Unstimulated_Replicate 1
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 36 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190521
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190521/suppl/GSM190521.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190522 | GPL201 |
|
Choroidal endothelial cell_Human 4_Unstimulated_Replicate 2
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 36 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190522
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190522/suppl/GSM190522.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190523 | GPL201 |
|
Choroidal endothelial cell_Human 4_Stimulated. LPS_Replicate 1
|
Choroidal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 36 years
Cell: choroidal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190523
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190523/suppl/GSM190523.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190524 | GPL201 |
|
Choroidal endothelial cell_Human 4_Stimulated. LPS_Replicate 2
|
Choroidal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 36 years
Cell: choroidal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 10/19/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190524
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190524/suppl/GSM190524.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190525 | GPL201 |
|
Retinal endothelial cell_Human 5_Unstimulated_Replicate 1
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 46 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190525
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190525/suppl/GSM190525.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190526 | GPL201 |
|
Retinal endothelial cell_Human 5_Unstimulated_Replicate 2
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 46 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190526
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190526/suppl/GSM190526.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190527 | GPL201 |
|
Retinal endothelial cell_Human 5_Stimulated.LPS_Replicate 1
|
Retinal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 46 years
Cell: retinal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190527
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190527/suppl/GSM190527.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190528 | GPL201 |
|
Retinal endothelial cell_Human 5_Stimulated.LPS_Replicate 2
|
Retinal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 46 years
Cell: retinal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190528
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190528/suppl/GSM190528.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190529 | GPL201 |
|
Choroidal endothelial cell_Human 5_Unstimulated_Replicate 1
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 46 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190529
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190529/suppl/GSM190529.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190530 | GPL201 |
|
Choroidal endothelial cell_Human 5_Unstimulated_Replicate 2
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: male
Age: 46 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190530
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190530/suppl/GSM190530.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190531 | GPL201 |
|
Choroidal endothelial cell_Human 5_Stimulated. LPS_Replicate 1
|
Choroidal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 46 years
Cell: choroidal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190531
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190531/suppl/GSM190531.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190532 | GPL201 |
|
Choroidal endothelial cell_Human 5_Stimulated. LPS_Replicate 2
|
Choroidal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: male
Age: 46 years
Cell: choroidal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 11/22/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190532
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190532/suppl/GSM190532.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190533 | GPL201 |
|
Retinal endothelial cell_Human 6_Unstimulated_Replicate 1
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 49 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190533
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190533/suppl/GSM190533.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190534 | GPL201 |
|
Retinal endothelial cell_Human 6_Unstimulated_Replicate 2
|
Retinal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 49 years
Cell: retinal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190534
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190534/suppl/GSM190534.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190535 | GPL201 |
|
Retinal endothelial cell_Human 6_Stimulated.LPS_Replicate 1
|
Retinal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: female
Age: 49 years
Cell: retinal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190535
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190535/suppl/GSM190535.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190536 | GPL201 |
|
Retinal endothelial cell_Human 6_Stimulated.LPS_Replicate 2
|
Retinal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: female
Age: 49 years
Cell: retinal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190536
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190536/suppl/GSM190536.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190537 | GPL201 |
|
Choroidal endothelial cell_Human 6_Unstimulated_Replicate 1
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 49 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190537
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190537/suppl/GSM190537.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190538 | GPL201 |
|
Choroidal endothelial cell_Human 6_Unstimulated_Replicate 2
|
Choroidal endothelial cell, medium alone, 4 hours
|
Gender: female
Age: 49 years
Cell: choroidal endothelial
Stimulus: medium alone
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190538
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190538/suppl/GSM190538.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190539 | GPL201 |
|
Choroidal endothelial cell_Human 6_Stimulated. LPS_Replicate 1
|
Choroidal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: female
Age: 49 years
Cell: choroidal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190539
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190539/suppl/GSM190539.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
GSM190540 | GPL201 |
|
Choroidal endothelial cell_Human 6_Stimulated. LPS_Replicate 2
|
Choroidal endothelial cell, lipopolysaccharide, 4 hours
|
Gender: female
Age: 49 years
Cell: choroidal endothelial
Stimulus: lipopolysaccharide
|
RNA input amount: 2ug; Hybridization date: 12/02/2005; Array lot: 4009762
|
| Sample_geo_accession | GSM190540
| | Sample_status | Public on May 25 2007
| | Sample_submission_date | May 18 2007
| | Sample_last_update_date | May 25 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | Once retinal and choroidal vascular endothelial cells had achieved confluence in the tissue culture dishes, the medium was changed to MCDB-131 supplemented with 5 % heat-inactivated FBS and endothelial growth factors. After overnight incubation, the medium was again changed as follows. For Humans 1, 2 and 3, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 3.0 x 106/mL tachyzoites in modified MCDB-131 medium or medium alone. For Humans 4, 5 and 6, separate dishes of choroidal and retinal endothelial cells were treated with 10 mL of either 100 ng/mL Escherichia coli 055:B55 LPS (Sigma-Aldrich) in modified MCDB-131 or medium alone. Plates were then incubated at 37 °C and 5 % CO2 for 4 hours.
| | Sample_growth_protocol_ch1 | Human eyes were obtained from 6 human cadaveric donors. No donor had a history of ocular disease. Death to isolation time varied from 8 to 19 hours. Cornea, if present, was excised, and iris was separated from the eye wall. Vitreous was removed from the posterior eyecup, and retina was separated from underlying choroid. Subsequently, choroid was separated from the eye wall and the retinal pigment epithelium was gently removed with a cotton bud. Choroid and retina were separately treated with graded solutions (0.25 to 3 mg/mL) of type II collagenase (Sigma-Aldrich, St Louis, MI) in HEPES-buffered MCDB-131 medium (Sigma-Aldrich; catalogue number 8537), supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1 mg/mL amphotericin B (GIBCO, Invitrogen, Grand Island, NY), at 37 °C and 5 % CO2, until the tissue was visibly digested. In the case of retina, digestion with type II collagenase was preceded by treatment with dispase (GIBCO, Invitrogen); an overnight incubation with 0.3 mg/mL dispase achieved optimal digestion. Subsequently, endothelial cells were purified from stromal cells using magnetic beads (Dynabeads) (Dynal, Invitrogen, Brown Deer, WI) coated with mouse monoclonal anti-human CD31 antibody (BD Pharmingen, San Diego, CA). Cells were cultured in MCDB-131 medium supplemented with 10 – 20 % FBS, endothelial growth factors and 1 mg/mL amphotericin B (Clonetics, Cambrex Bioscience, Walkerville, MD; EGM-2 SingleQuot catalogue number cc-4176), but without addition of gentamicin, hydrocortisone and FBS), at 37 °C and 5 % CO2, and passed with the use of 0.05 % trypsin (GIBCO, Invitrogen). Cultured cells were used at passage 5 or less. Immediately prior to treatment, retinal and choroidal vascular endothelial cells were grown to confluence simultaneously in separate 10 cm polystyrene tissue culture dishes.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Following the treatment, cell supernatants were removed. A 0.6 mL aliquot of RLT lysis buffer (Qiagen, Valencia, CA) was evenly spread across the cells of each plate, and plates were immediately stored at –80 °C. Upon thawing, cell lysates were collected using a cell scraper and immediately homogenized using Qiashredder spin columns (Qiagen). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions, including the optional on-column DNase treatment. RNA was eluted from the column using nuclease-free water (Ambion, Austin, TX), with 2 rounds of 50 ml yielding a total elute of 100 ml. To concentrate the RNA, it was mixed in a solution containing 10 ml of 2 M sodium acetate at pH 4.0 (Stratagene, Cedar Creek, TX) and 110 ml of 100 % isopropanol, and precipitated overnight at –20 °C. RNA pellets were washed using graded solutions of ethanol, dried in air, and subsequently raised in 10 µl of nuclease-free water. Quantity and purity of the RNA were determined by spectrophotometry.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. Biotinylated cRNA target was prepared according to the standard Affymetrix one-cycle cDNA protocol from 1-2 ug total RNA (Expression Analysis Technical Manual, Ver. 701021 Rev.5). Standard pre-mixed Affymetrix hybridization controls (bioB, bioC, bioD, and cre at 1.5, 5, 25, and 100 pM concentrations, respectively) and Control Oligo B2 were added directly to the hybridization cocktail according to manufacturer's protocol.
| | Sample_hyb_protocol | Following fragmentation, 6.5 ug of cRNA were hybridized for 16 hrs at 45 °C on Human Genome Focus GeneChip Arrays. GeneChip arrays were washed and stained in the Affymetrix Fluidics Station 400 for 193JS Set#1. 193JS Set#2-3 and 245JS Sets#1-3 were washed and stained in the Affymetrix Fluidics Station 450.
| | Sample_scan_protocol | GeneChip arrays were scanned using the Affymetrix GeneChip Scanner 3000 and GCOS version 1.2 software (Affymetrix), yielding cell fluorescence intensity (.CEL) files.
| | Sample_data_processing | Array data were preprocessed and normalized as two separate sets. Sample assay names with the suffix, 193JS were grouped as set1 and all sample names with the suffix, 245JS were grouped as set2. Data preprocessing and normalization were performed with the “affy“ and “gcrma” packages of the Bioconductor project (http://www.bioconductor.org) that run above the R statistical language environment. .CEL files were imported into the R environment. Perfect match (PM) probe data were corrected for background noise using the GeneChip robust multi-array analysis (GCRMA). Corrected PM probe data were normalized with the algorithm based on rank invariant probes. Gene expression values were determined using a linear model estimated by the median polish algorithm.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Justine,,Smith
| | Sample_contact_email | smithjus@ohsu.edu
| | Sample_contact_phone | 503-494-5023
| | Sample_contact_fax | 503-494-6875
| | Sample_contact_department | Casey Eye Institute
| | Sample_contact_institute | Oregon Health & Science University
| | Sample_contact_address | 3181 SW Sam Jackson Park Road, Biomedical Research Building, Level 2
| | Sample_contact_city | Portland
| | Sample_contact_state | OR
| | Sample_contact_zip/postal_code | 97239
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM190nnn/GSM190540/suppl/GSM190540.CEL.gz
| | Sample_series_id | GSE7850
| | Sample_data_row_count | 8793
| | |
|
| |
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