8 protocols
ID_REF = VALUE = quantile normalization Detection Pval =
Data was quantile-normalized in BeadStudio by Yale Center for Genome Analysis (normalized data provided here). Following initial normalization, additional adjustment for between-donor variation was made. For each donor, each gene’s expression value was normalized by that gene’s median expression value in that donor. Low expression genes were filtered out, and the remaining expression data were log2-transformed. Differentially expressed genes (vs. untreated cells) were selected using a two-tail unpaired t-test with a False Discovery Rate-corrected threshold of 0.05
Standard Illumina scanning protocol, performed by Yale Center for Genome Analysis
Standard Illumina hybridization protocol, performed by Yale Center for Genome Analysis
Preparation of labeled cRNA for hybridization onto Illumina BeadChips followed the recommended Illumina protocol using a TotalPrep RNA Amplification kit (Applied Biosystems), performed by the Yale Center for Genome Analysis
Total RNA was isolated from DCs using a Qiagen Mini RNeasy kit or a Qiagen BioRobot Universal with RNeasy 96 kit, with on-column DNase treatment, according to the manufacturer’s protocol. RNA was quantified using a ND-1000 spectrophotometer (NanoDrop)
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll density gradient centrifugation (Histopaque, Sigma-Aldrich). CD14+ monocytes were then immunomagnetically purified from PBMCs using a MACS CD14 isolation kit (Miltenyi Biotec). CD14+ monocytes were then differentiated into naïve, immature DCs by 5-day incubation in DC growth media [RPMI medium 1640 (Gibco), 10% fetal calf serum (Hyclone), 2mM of L-glutamine, 100 U/ml penicillin (Invitrogen), and 100 U/ml streptomycin (Invitrogen)] supplemented with 500 U/ml hGM-CSF (Peprotech) and 1000 U/ml hIL-4 (Peprotech) at 37°C. DCs were then re-suspended in cytokine-free DC growth media (106 cells/ml) and incubated for 1 day at 37°C. DCs from randomly selected buffy coats were assayed for surface CD11c expression (a dendritic cell marker) as a quality control for cell uniformity. All assayed DCs homogeneously expressed surface CD11c.
DCs were treated with 10 ng/ml of hIL-6 (Peprotech) or 10 ng/ml of hIL-10 (Peprotech)