Search results for the GEO ID: GSE18391  |
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GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM458608 | GPL570 |
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Uninduced
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Adipose-derived stromal/stem cells (ASC)
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protocol: uninduced (control)
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Adipose-derived stromal/stem cells (ASC) capable of multipotential differentiation can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our recent experience isolating and immunophenotypically characterizing ASCs from >60 human subjects
Total RNA was isolated from ASCs obtained from three individual donors and maintained under uninduced or adipogenic induced culture conditions for 9 days following confluence. The RNA was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), cDNA synthesized using a Superscript cDNA Synthesis kit (Invitrogen, Carlsbad, CA) and a T7-(dT)24 primer. Biotinylated cRNA was transcribed in vitro using a GeneChip IVT Labeling kit (Affymetrix, Santa Clara, CA), purified using a GeneChip Sample Cleanup Module, incubated in fragmentation buffer (200 mM Tris-acetate pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94oC for 35 min, chilled on ice, and hybridized to a Human U133 Plus 2.0 Array of ~47,400 transcripts and variants, including 38,500 well-characterized genes. Arrays were incubated at 45oC for 16 hrs rotating at 60 rpm, washed, stained at 25oC for 10 min with 10 μg/ml streptavidin R phycoerythrin (Vector Laboratories, Burlingame, CA), stained at 25oC for 10 min (X2) with 3 μg/ml biotinylated goat anti-streptavidin antibody (Vector Laboratories), washed, and scanned using a GeneChip Scanner 3000. Pixel intensities were measured, expression signals were globally scaled to a target intensity value of 2500, and features extracted using GeneChip Operating Software v1.2 (Affymetrix). Data mining and statistical analyses were performed with Data Mining Tool v.3 (Affymetrix) algorithms. The absolute call (present, marginal, absent) of each gene expression, the direction of change, and the fold change were identified with the above software.
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| Sample_geo_accession | GSM458608
| | Sample_status | Public on Oct 01 2010
| | Sample_submission_date | Oct 02 2009
| | Sample_last_update_date | Oct 05 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_biomaterial_provider_ch1 | Pennington Biomedical Research Center
| | Sample_treatment_protocol_ch1 | Trypsin harvested ASCs were washed with PBS three times and aliquots of 105 cells were incubated with phycoerythrin conjugated monoclonal antibodies directed against CD29 (eBioscience Cat # 12-0297) CD34 (Becton Dickinson Cat # 348057), CD45 (eBioscience Cat # 12-0459), CD73 (BD Pharmingen Cat # 550257), CD90 (BD Pharmingen Cat # 55596), or IgG1κ control (BD Pharmingen Cat # 555749) or FITC conjugated monoclonal antibodies directed against CD44 (BD Cat # 348057) or IgG1κ control (BD Pharmingen Cat # 554679) for 20 min on ice before being washed with PBS supplemented with 1% BSA three times and fixed in 1% formaldehyde overnight at 4oC. For each sample, 105 events were collected on a Becton Dickinson FACScaliber flow cytometer using CELLQuest acquisition software (Becton Dickinson) and analyzed using Flow Jo software (Tree Star). This antibody panel was selected, in part, based on the ISCT position paper on the criteria for defining MSCs (17). Confluent cultures of ASC (P1) were induced with Adipogenic Differentiation Medium (DMEM/Hams F-12, 3% FBS, 1% antibiotic/antimycotic, 0.5 mM isobutylmethylxanthine, 33 µM biotin, 17 µM pantothenate, 5 μM rosiglitazone (AK Scientific, Mountain View, CA), 1 µM dexamethasone, 1 μM insulin) for three days before being converted to Adipocyte Maintenance Medium (identical to Adipogenic Differentiation Medium without isobutylmethylxanthine and rosiglitazone). Cells were maintained for 9 days before fixation and Oil Red O staining. In selected experiments, samples of uninduced and adipogenic induced ASC were harvested for total RNA isolation at days 0, 3, 6, and 9 of differentiation. Confluent cultures of ASCs were converted to Osteogenic Medium (DMEM/Hams F-12 or DMEM, 10% FBS, 1% antibiotic/antimycotic, 10 mM β-glycerophosphate, 50 µg/ml sodium 2-phosphate ascorbate, 10-8 M dexamethasone) and maintained in culture for 9-12 days with medium changes every third day. The cultures were rinsed three times with 150 mM NaCl, fixed in 70% ethanol, and stained with Alizarin Red.
| | Sample_growth_protocol_ch1 | ASC were isolated from human fresh human subcutaneous adipose lipoaspirate according to published methods with some minor modifications (9,15). The lipoaspirate tissue was extensively washed with warm phosphate buffer solution to remove erythrocytes and then digested in PBS supplemented with 0.1% Collagenase of Type I (Worthington Biochemical Corporation), 1% BSA, and 2 mM CaCl2 for one hour at 37oC. Following room temperature centrifugation at 300 X g and resuspension in Stromal Medium (DMEM/Hams F-12 Medium supplemented with 10% FBS (Hyclone, Logan UT) and 1% antibiotic/antimycotic, the stromal vascular pellet was plated at a density of 35 ml of lipoaspirate digest per T175 flasks (0.2 ml per sq cm). After 24 hrs of incubation at 37oC, 5% CO2, the adherent cells were washed with warm PBS and maintained in Stromal Medium until 80-90% confluent. The adherent population was harvested by digestion with trypsin (0.05%)/EDTA (1 mM) at 37oC for five minutes, washed in Stromal Medium and replated at 5 X 103 ASC per sq cm (“Passage 1” or “P1”) or used in flow cytometric analyses or cryopreserved for future use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was isolated from ASC cultures using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. The total RNA (2 µg) was incubated in a 20 µl volume with Moloney Murine Leukemia Virus Reverse Transcriptase, dNTP, and olig-dT.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Arrays were incubated at 45oC for 16 hrs rotating at 60 rpm, washed, stained at 25oC for 10 min with 10 μg/ml streptavidin R phycoerythrin (Vector Laboratories, Burlingame, CA), stained at 25oC for 10 min (X2) with 3 μg/ml biotinylated goat anti-streptavidin antibody (Vector Laboratories), washed, and scanned using a GeneChip Scanner 3000.
| | Sample_hyb_protocol | Arrays were incubated at 45oC for 16 hrs rotating at 60 rpm, washed, stained at 25oC for 10 min with 10 μg/ml streptavidin R phycoerythrin (Vector Laboratories, Burlingame, CA), stained at 25oC for 10 min (X2) with 3 μg/ml biotinylated goat anti-streptavidin antibody (Vector Laboratories), washed, and scanned using a GeneChip Scanner 3000.
| | Sample_scan_protocol | Pixel intensities were measured, expression signals were globally scaled to a target intensity value of 2500, and features extracted using GeneChip Operating Software v1.2 (Affymetrix).
| | Sample_data_processing | Probe set signal intensities calculated with GeneChip Operating Software v1.2 (Affymetrix).
| | Sample_data_processing | Data mining and statistical analyses were performed with Data Mining Tool v.3 (Affymetrix) algorithms.
| | Sample_platform_id | GPL570
| | Sample_contact_name | Andrey,,Ptitsyn
| | Sample_contact_email | andrey.ptitsyn@colostate.edu
| | Sample_contact_phone | 970-491-0878
| | Sample_contact_department | Center for Bioinformatics
| | Sample_contact_institute | Colorado State University
| | Sample_contact_address | 1682 Canpus Delivery
| | Sample_contact_city | Fort Collins
| | Sample_contact_state | CO
| | Sample_contact_zip/postal_code | 80523
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM458nnn/GSM458608/suppl/GSM458608.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM458nnn/GSM458608/suppl/GSM458608.CHP.gz
| | Sample_series_id | GSE18391
| | Sample_data_row_count | 54675
| | |
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GSM458609 | GPL570 |
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Induced
|
Adipose-derived stromal/stem cells (ASC)
|
protocol: adipogenic induced
|
Adipose-derived stromal/stem cells (ASC) capable of multipotential differentiation can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our recent experience isolating and immunophenotypically characterizing ASCs from >60 human subjects
Total RNA was isolated from ASCs obtained from three individual donors and maintained under uninduced or adipogenic induced culture conditions for 9 days following confluence. The RNA was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), cDNA synthesized using a Superscript cDNA Synthesis kit (Invitrogen, Carlsbad, CA) and a T7-(dT)24 primer. Biotinylated cRNA was transcribed in vitro using a GeneChip IVT Labeling kit (Affymetrix, Santa Clara, CA), purified using a GeneChip Sample Cleanup Module, incubated in fragmentation buffer (200 mM Tris-acetate pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94oC for 35 min, chilled on ice, and hybridized to a Human U133 Plus 2.0 Array of ~47,400 transcripts and variants, including 38,500 well-characterized genes. Arrays were incubated at 45oC for 16 hrs rotating at 60 rpm, washed, stained at 25oC for 10 min with 10 μg/ml streptavidin R phycoerythrin (Vector Laboratories, Burlingame, CA), stained at 25oC for 10 min (X2) with 3 μg/ml biotinylated goat anti-streptavidin antibody (Vector Laboratories), washed, and scanned using a GeneChip Scanner 3000. Pixel intensities were measured, expression signals were globally scaled to a target intensity value of 2500, and features extracted using GeneChip Operating Software v1.2 (Affymetrix). Data mining and statistical analyses were performed with Data Mining Tool v.3 (Affymetrix) algorithms. The absolute call (present, marginal, absent) of each gene expression, the direction of change, and the fold change were identified with the above software.
|
| Sample_geo_accession | GSM458609
| | Sample_status | Public on Oct 01 2010
| | Sample_submission_date | Oct 02 2009
| | Sample_last_update_date | Oct 05 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_biomaterial_provider_ch1 | Pennington Biomedical Research Center
| | Sample_treatment_protocol_ch1 | Trypsin harvested ASCs were washed with PBS three times and aliquots of 105 cells were incubated with phycoerythrin conjugated monoclonal antibodies directed against CD29 (eBioscience Cat # 12-0297) CD34 (Becton Dickinson Cat # 348057), CD45 (eBioscience Cat # 12-0459), CD73 (BD Pharmingen Cat # 550257), CD90 (BD Pharmingen Cat # 55596), or IgG1κ control (BD Pharmingen Cat # 555749) or FITC conjugated monoclonal antibodies directed against CD44 (BD Cat # 348057) or IgG1κ control (BD Pharmingen Cat # 554679) for 20 min on ice before being washed with PBS supplemented with 1% BSA three times and fixed in 1% formaldehyde overnight at 4oC. For each sample, 105 events were collected on a Becton Dickinson FACScaliber flow cytometer using CELLQuest acquisition software (Becton Dickinson) and analyzed using Flow Jo software (Tree Star). This antibody panel was selected, in part, based on the ISCT position paper on the criteria for defining MSCs (17). Confluent cultures of ASC (P1) were induced with Adipogenic Differentiation Medium (DMEM/Hams F-12, 3% FBS, 1% antibiotic/antimycotic, 0.5 mM isobutylmethylxanthine, 33 µM biotin, 17 µM pantothenate, 5 μM rosiglitazone (AK Scientific, Mountain View, CA), 1 µM dexamethasone, 1 μM insulin) for three days before being converted to Adipocyte Maintenance Medium (identical to Adipogenic Differentiation Medium without isobutylmethylxanthine and rosiglitazone). Cells were maintained for 9 days before fixation and Oil Red O staining. In selected experiments, samples of uninduced and adipogenic induced ASC were harvested for total RNA isolation at days 0, 3, 6, and 9 of differentiation. Confluent cultures of ASCs were converted to Osteogenic Medium (DMEM/Hams F-12 or DMEM, 10% FBS, 1% antibiotic/antimycotic, 10 mM β-glycerophosphate, 50 µg/ml sodium 2-phosphate ascorbate, 10-8 M dexamethasone) and maintained in culture for 9-12 days with medium changes every third day. The cultures were rinsed three times with 150 mM NaCl, fixed in 70% ethanol, and stained with Alizarin Red.
| | Sample_growth_protocol_ch1 | ASC were isolated from human fresh human subcutaneous adipose lipoaspirate according to published methods with some minor modifications (9,15). The lipoaspirate tissue was extensively washed with warm phosphate buffer solution to remove erythrocytes and then digested in PBS supplemented with 0.1% Collagenase of Type I (Worthington Biochemical Corporation), 1% BSA, and 2 mM CaCl2 for one hour at 37oC. Following room temperature centrifugation at 300 X g and resuspension in Stromal Medium (DMEM/Hams F-12 Medium supplemented with 10% FBS (Hyclone, Logan UT) and 1% antibiotic/antimycotic, the stromal vascular pellet was plated at a density of 35 ml of lipoaspirate digest per T175 flasks (0.2 ml per sq cm). After 24 hrs of incubation at 37oC, 5% CO2, the adherent cells were washed with warm PBS and maintained in Stromal Medium until 80-90% confluent. The adherent population was harvested by digestion with trypsin (0.05%)/EDTA (1 mM) at 37oC for five minutes, washed in Stromal Medium and replated at 5 X 103 ASC per sq cm (“Passage 1” or “P1”) or used in flow cytometric analyses or cryopreserved for future use.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was isolated from ASC cultures using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. The total RNA (2 µg) was incubated in a 20 µl volume with Moloney Murine Leukemia Virus Reverse Transcriptase, dNTP, and olig-dT.
| | Sample_label_ch1 | biotin
| | Sample_label_protocol_ch1 | Arrays were incubated at 45oC for 16 hrs rotating at 60 rpm, washed, stained at 25oC for 10 min with 10 μg/ml streptavidin R phycoerythrin (Vector Laboratories, Burlingame, CA), stained at 25oC for 10 min (X2) with 3 μg/ml biotinylated goat anti-streptavidin antibody (Vector Laboratories), washed, and scanned using a GeneChip Scanner 3000.
| | Sample_hyb_protocol | Arrays were incubated at 45oC for 16 hrs rotating at 60 rpm, washed, stained at 25oC for 10 min with 10 μg/ml streptavidin R phycoerythrin (Vector Laboratories, Burlingame, CA), stained at 25oC for 10 min (X2) with 3 μg/ml biotinylated goat anti-streptavidin antibody (Vector Laboratories), washed, and scanned using a GeneChip Scanner 3000.
| | Sample_scan_protocol | Pixel intensities were measured, expression signals were globally scaled to a target intensity value of 2500, and features extracted using GeneChip Operating Software v1.2 (Affymetrix).
| | Sample_data_processing | Probe set signal intensities calculated with GeneChip Operating Software v1.2 (Affymetrix).
| | Sample_data_processing | Data mining and statistical analyses were performed with Data Mining Tool v.3 (Affymetrix) algorithms.
| | Sample_platform_id | GPL570
| | Sample_contact_name | Andrey,,Ptitsyn
| | Sample_contact_email | andrey.ptitsyn@colostate.edu
| | Sample_contact_phone | 970-491-0878
| | Sample_contact_department | Center for Bioinformatics
| | Sample_contact_institute | Colorado State University
| | Sample_contact_address | 1682 Canpus Delivery
| | Sample_contact_city | Fort Collins
| | Sample_contact_state | CO
| | Sample_contact_zip/postal_code | 80523
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM458nnn/GSM458609/suppl/GSM458609.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM458nnn/GSM458609/suppl/GSM458609.CHP.gz
| | Sample_series_id | GSE18391
| | Sample_data_row_count | 54675
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