10 protocols
AccessionType
bioassay_data_transformation
Title: CHP Analysis. Description:
specified_biomaterial_action
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.
hybridization
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.
grow
Sample was cultured first in Stem Span serum free and later in RPMI+10%FCS+2mM L-glutamine.
specified_biomaterial_action
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.
labeling
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.
nucleic_acid_extraction
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).
specified_biomaterial_action
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.
specified_biomaterial_action
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.
image_acquisition
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.