Affymetrix GeneChip Human Genome U95Av2 [HG_U95Av2]

Transcriptional profiling identifies Id2 function in dendritic cell development.

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Dendritic Cell differentiation -  CD molecule cluster follow up: The data files associated to this experiment show the gene expression levels for a subset of 152 transcripts (out of 12626 genes represented on Affymetrix Genechip HG_U95Av2) representing CD molecules specifically expressed in Dendritic Cells (DC) as assessed by the 9 conditions tested. Another subset of genes, corresponding to a cluster of Transcription regulators is available from  E-MEXP-2 experiment.

Transcription profiling of dendritic cells under 9 conditions

"Dendritic Cell differentiation -  CD molecule cluster follow up: The data files associated to this experiment show the gene expression levels for a subset of 152 transcripts (out of 12626 genes represented on Affymetrix Genechip HG_U95Av2) representing CD molecules specifically expressed in Dendritic Cells (DC) as assessed by the 9 conditions tested. Another subset of genes, corresponding to a cluster of Transcription regulators is available from  E-MEXP-2 experiment."

Dendritic Cell differentiation -  CD molecule cluster follow up: The data files associated to this experiment show the gene expression levels for a subset of 152 transcripts (out of 12626 genes represented on Affymetrix Genechip HG_U95Av2) representing CD molecules specifically expressed in Dendritic Cells (DC) as assessed by the 9 conditions tested. Another subset of genes, corresponding to a cluster of Transcription regulators is available from  E-MEXP-2 experiment.


P-AFFY-7

Protocol:236

Total RNA was extracted using RNeasy Midikit (Qiagen, Hilden, Germany) using the maufacturer's buffers and protocols. An additional DNAse digestion with RNAse free DNAse (Qiagen) was performed. First and second strand cDNA synthesis were done from 7 ug total RNA with the SuperScript Choice system (Gibco BRL) and a T7-(dT)24 Primer (Biotez, Berlin, Germany).

Protocol:237

In vitro transcription employing double stranded cDNA as template and biotinylated UTP and CTP (Perkin Elmer/ENZO) as label using the MEGAscript kit from Ambion according to the manufacturer's instructions.

Protocol:241

Arrays were prehybridized with 200 ul MES hybridization buffer for 10 minutes at 45C with rotation (60U/min). After removing the prehybridization solution, 10 ug of labelled cRNA in 200 ul MES hybridization buffer were applied to the array thereby leaving a small air bubble. Arrays were incubated overnight at 45C with rotation (60U/min) in the hybridization chamber.

Laser was warmed up for 15 min. before scanning. Stained arrays (room temp) were inserted into the scanner and scanned 2x at 570 nm with 6 um pixel value.

Protocol:258

Mononuclear cells from human cord blood were recovered by Ficoll-Hypaque gradient centrifugation (density 1.077 g/ml) and CD34+ cells were isolated using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany). CD34+ cells were obtained by two cycles of immunomagnetic bead selection and selected cells were analyzed by flow cytometry. Purity was 83-90%.  CD34+ cells (0.3-0.5 million cells/ml) were cultured in StemSpan serum free medium (StemCell Technologies Inc., Vancouver, Canada) with 100 ng/ml stem cell factor (SCF), 50 ng/ml Flt3 ligand (FL), 20 ng/ml thrombopoietin (Tpo) and 10 ng/ml IL-6/soluble IL-6 receptor fusion protein (hyper-IL-6). Growth factors were added every 2 days and cells were maintained at 1 million cells/ml cell density for 12 days. 

Protocol:261

DC were obtained from CD34+ progenitor cells of human cord blood by employing a two-step amplification/differentiation protocol. Mononuclear cells (MNC) were recovered by Ficoll-Hypaque gradient centrifugation and CD34+ cells were isolated using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany). CD34+ cells were obtained by two cycles of immunomagnetic bead selection and selected cells were analyzed by flow cytometry. Purity was 83-90%.CD34+ cells (0.3-0.5 million cells/ml) were cultured in StemSpan serum free medium (StemCell Technologies Inc., Vancouver, Canada) with 100 ng/ml stem cell factor (SCF), 50 ng/ml Flt3 ligand (FL), 20 ng/ml thrombopoietin (Tpo) and 10 ng/ml IL-6/soluble IL-6 receptor fusion protein (hyper-IL-6). Growth factors were added every 2 days and cells were maintained at 1 million cells/ml cell density.  After 12 days of culture progenitor cells were induced to differentiate into DC in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin and streptomycin (GIBCO Invitrogen Corporation, Paisley, UK) and 500 U/ml GM-CSF, 500 U/ml IL-4 for 6 days (0.5 million cells/ml). Every 2 days growth factors were added and cells were maintained at 0.5 million cells/ml cell density. 

Protocol:262

Protocol:264

DC were obtained from CD34+ progenitor cells of human cord blood by employing a two-step amplification/differentiation protocol. Mononuclear cells (MNC) were recovered by Ficoll-Hypaque gradient centrifugation and CD34+ cells were isolated using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany). CD34+ cells were obtained by two cycles of immunomagnetic bead selection and selected cells were analyzed by flow cytometry. Purity was 83-90%.CD34+ cells (0.3-0.5 million cells/ml) were cultured in StemSpan serum free medium (StemCell Technologies Inc., Vancouver, Canada) with 100 ng/ml stem cell factor (SCF), 50 ng/ml Flt3 ligand (FL), 20 ng/ml thrombopoietin (Tpo) and 10 ng/ml IL-6/soluble IL-6 receptor fusion protein (hyper-IL-6). Growth factors were added every 2 days and cells were maintained at 1 million cells/ml cell density.  After 12 days of culture progenitor cells were induced to differentiate into DC in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin and streptomycin (GIBCO Invitrogen Corporation, Paisley, UK) and 500 U/ml GM-CSF, 500 U/ml IL-4 for 6 days (0.5 million cells/ml). Every 2 days growth factors were added and cells were maintained at 0.5 million cells/ml cell density. 10 ng/ml TNFa was added at day 6 for an additional 24 h to induce DC maturation.

Protocol:468

Sample was cultured first in Stem Span serum free and later in RPMI+10%FCS+2mM L-glutamine.

Transcription profiling of dendritic cells under 9 conditions

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