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Search results for the GEO ID: GSE8334

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GSM206416

GPL339

VDRwt DC - untreated

VDRwt 2-step DC - untreated

Mouse strain: vitamin D3 receptor mutant (mut) and wild-type (wt) (see Erben, R. G., Soegiarto, D. W., Weber, K., Zeitz, U., Lieberherr, M., Gniadecki, R., Moller, G., Adamski, J. and Balling, R., Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D. Mol Endocrinol 2002. 16: 1524-1537. Gender: male (mut and wt); Age: 9 weeks (mut and wt); Source tissue: bone marrow; Cell type: in vitro generated dendritic cells.

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206416
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206416/suppl/GSM206416.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206416/suppl/GSM206416.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206416/suppl/GSM206416.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206416/suppl/GSM206416.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206416/suppl/GSM206416.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206416/suppl/GSM206416.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

GSM206648

GPL339

VDRwt DC - VD3

VDRwt 2-step DC - VD3 pre-treated 30 min

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206648
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206648/suppl/GSM206648.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206648/suppl/GSM206648.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206648/suppl/GSM206648.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206648/suppl/GSM206648.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206648/suppl/GSM206648.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206648/suppl/GSM206648.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

GSM206649

GPL339

VDRwt DC - TNFa

VDRwt 2-step DC - TNFa 4h

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206649
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206649/suppl/GSM206649.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206649/suppl/GSM206649.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206649/suppl/GSM206649.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206649/suppl/GSM206649.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206649/suppl/GSM206649.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206649/suppl/GSM206649.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

GSM206650

GPL339

VDRwt DC - VD3+TNFa

VDRwt 2-step DC - VD3 pre-treated 30 min, TNFa 4h

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206650
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206650/suppl/GSM206650.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206650/suppl/GSM206650.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206650/suppl/GSM206650.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206650/suppl/GSM206650.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206650/suppl/GSM206650.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206650/suppl/GSM206650.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

GSM206652

GPL339

VDRmut DC - untreated

VDRmut 2-step DC - untreated

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206652
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206652/suppl/GSM206652.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206652/suppl/GSM206652.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206652/suppl/GSM206652.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206652/suppl/GSM206652.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206652/suppl/GSM206652.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206652/suppl/GSM206652.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

GSM206653

GPL339

VDRmut DC - VD3

VDRmut 2-step DC - VD3 pre-treated 30 min

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206653
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206653/suppl/GSM206653.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206653/suppl/GSM206653.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206653/suppl/GSM206653.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206653/suppl/GSM206653.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206653/suppl/GSM206653.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206653/suppl/GSM206653.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

GSM206654

GPL339

VDRmut DC - TNFa

VDRmut 2-step DC - TNFa 4h

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206654
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206654/suppl/GSM206654.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206654/suppl/GSM206654.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206654/suppl/GSM206654.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206654/suppl/GSM206654.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206654/suppl/GSM206654.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206654/suppl/GSM206654.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

GSM206656

GPL339

VDRmut DC - VD3+TNFa

VDRmut 2-step DC - VD3 pre-treated 30 min, TNFa 4h

cRNA labelling, array hybridisation, washing and scanning performed at the Microarray Facility of the Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Sample_geo_accession	GSM206656
Sample_status	Public on Dec 31 2012
Sample_submission_date	Jun 29 2007
Sample_last_update_date	Dec 31 2012
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Kindly provided by A. Lengeling, German Research Centre for Biotechnology, GBF, Braunschweig, Germany.
Sample_treatment_protocol_ch1	Dendritic cells (DC) from VDRmut and control VDRwt mice were generated as described in the Growth Protocol section. On day 7 differentiation DC were pre-treated for 30 min with 1a,25-dihydroxyvitamin D3 (VD3) at 10^-6M, or left untreated. TNFa (50 ng/ml) was added where appropriate, and cells incubated further for 4 h.
Sample_growth_protocol_ch1	Dendritic cells (DC) were generated as previously described (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562).
Sample_growth_protocol_ch1	Bone marrow was prepared from hind legs of a 9-weeks old (male) VDRmut and VDRwt control mice. Briefly, homogenised cell suspension was diluted to 2x10^6 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin sulphate, 50 uM b-mercaptoethanol. Medium was supplemented with the following cytokines/growth factors: 20 U/ml murine SCF, 5 ng/ml hyperIL-6, 25 ng/ml Flt-3L, 40ng/ml human IGF-1 long range, 10^-6 uM dexamethasone and 25 U/ml murine GM-CSF. Cells were then cultured at 37°C in 5% CO2.
Sample_growth_protocol_ch1	After 3 days in culture cells were applied to density gradient centrifugation using Ficoll-Hypaque (1,077g/ml) to remove dead cells and debris and to recover factor-dependent progenitor cells. Cells were washed once in complete RPMI medium and adjusted to 2x10^6 cells/ml; above listed growth factors were added again. Medium was renewed every second day and cells were adjusted to 2x10^6 cells/ml, supplementing with growth factors over again. (Cells were maintained in these conditions for 7 days.)
Sample_growth_protocol_ch1	At day 7 of culture differentiation of DC from the proliferating progenitor cells was initiated. Cells were washed once in RPMI medium and adjusted to 2x10^6 cells/ml. Cells were seeded in new dishes and murine GM-CSF was added at final concentration of 250U/ml. Medium was renewed every second day, supplementing with 250 U/ml GM-CSF. Since cells typically carry on proliferating in the first 3-4 days of differentiation, they were adjusted to the concentration of 2x10^6 cells/ml until onset of proliferation during every medium change. After 7 days of differentiation from progenitor cells a homogenous population of DC (in suspension; beside strongly adherent macrophages) developed. Non-adherent DC were used for the experimental treatment.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	Total RNA was isolated with RNeasy Midi Kit including DNase digestion (Qiagen).
Sample_label_ch1	Phycoerythrin (PE)
Sample_label_protocol_ch1	5 ug of total RNA was used to generate cRNA using a modified protocol from the Expression Analysis Technical Manual (Affymetrix).
Sample_label_protocol_ch1	RNA was concentrated to >0.5 ug/ul by acid-ethanol precipitation. For the first-strand cDNA synthesis a T7-oligo(dT) primer was firstly hybridised to 8 ug of total RNA by denaturation for 10 min at 70°C, then quick-chilling on ice, followed by incubation for 2 min at 42°C. The reverse transcription reaction was performed using 200U SuperScript RT (Invitrogen) per sample for 1 h at 42°C. The second-strand cDNA synthesis reaction was carried out by firstly incubating the single-stranded cDNA with 10U of DNA ligase, 40U of DNA Polimerase I and 2U of RNase H for 2 h at 16°C, and then additionally for 5 min at 16°C with T4 DNA Polymerase in order to enhance the efficiency of the second-strand synthesis. cDNA was cleaned-up using the QIAquick Spin PCR Purification Kit (Qiagen). Buffer PB was used for binding: this allows removal of fragments smaller than 100 bp, i.e. T7-oligo(dT) primers. DNA was eluted with elution buffer (EB). Double-stranded cDNA was then concentrated again by acid-ethanol precipitation.
Sample_label_protocol_ch1	Synthesis of biotin-labelled cRNA was conducted using T7 Mega Script Kit (Ambion). cDNA template was incubated with dNTPs in the presence of Bio-11-CTP and Bio-11-UTP (Perkin Elmer) with the T7 RNA Polymerase. Reaction incubated for 6 h at 37°C. cRNA was purified using RNeasy Mini Kit (Qiagen). cRNA was then fragmented using the fragmentation buffer.
Sample_label_protocol_ch1	Further processing of probes was performed according to the recommendations of the Expression Analysis Technical Manual (Affymetrix).
Sample_hyb_protocol	Chip Type: MOE430A; Chip Lot: 3001946; Protocol: EukGE-WS2; Wash A1 Temperature (C): 25; Number of Wash A1 Cycles: 10; Mixes per Wash A1 Cycle: 2; Wash B Temperature (C): 50; Number of Wash B Cycles: 4; Mixes per Wash B Cycle: 15; Stain Temperature (C): 25; First Stain Time (seconds): 600; Wash A2 Temperature (C): 25; Number of Wash A2 Cycles: 10; Mixes per Wash A2 Cycle: 4; Second Stain Time (seconds): 600; Third Stain Time (seconds): 600; Wash A3 Temperature (C): 30; Number of Wash A3 Cycles: 15; Mixes per Wash A3 Cycle: 4; Holding Temperature (C): 25; Station: 1; Module: 2.
Sample_scan_protocol	Pixel Size: 3; Filter: 570; Number of Scans: 2; Scanner Type: HP.
Sample_data_processing	Scanned GeneChip *.DAT files were processed by the Affymetrix Microarray Suite 5.0.1 analysis software. Scaling: All probe sets, TGT Value: 200; Normalisation: All probe sets.
Sample_platform_id	GPL339
Sample_contact_name	Mykola,,Goncharenko
Sample_contact_email	nick.goncharenko@berlin.de
Sample_contact_phone	+492418080763
Sample_contact_fax	+492418082442
Sample_contact_department	Institute for Biomedical Technology - Cell Biology
Sample_contact_institute	IBMT - Cell Biology, University Hospital, RWTH Aachen
Sample_contact_address	Pauwelstrasse 30
Sample_contact_city	Aachen
Sample_contact_zip/postal_code	52074
Sample_contact_country	Germany
Sample_contact_web_link	http://molcell.de
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206656/suppl/GSM206656.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206656/suppl/GSM206656.CHP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206656/suppl/GSM206656.EXP.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206656/suppl/GSM206656.RPT.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206656/suppl/GSM206656.TIF.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM206nnn/GSM206656/suppl/GSM206656.TXT.gz
Sample_series_id	GSE8334
Sample_data_row_count	22690

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