gender: M disease: Healthy Control stimulation: Unstimulated batch: batch2
Treatment protocol
cells are untreated
Growth protocol
Peripheral venous blood samples were collected into heparin (5 U/ml). Mononuclear cells were isolated by differential centrifugation (900 g, 30 mins, 20°C) over Lymphoprep® (Axis-Shield, Oslo, Norway) and washed twice with sterile phosphate-buffered saline (PBS) (GIBCO, Paisley, UK) at 300 g (5 mins, 20°C). Cells were resuspended in 10 ml RPMI (Invitrogen, Paisley, UK) supplemented with 100 U/ml of penicillin (GIBCO) and 100 mg/ml streptomycin (GIBCO) and 20 mM Hepes buffer (Sigma-Aldridge, Poole, UK) (RPMI), and plated at a density of approximately 5×106 cells/ml in 8cm2 NunclonTM Surface tissue culture dishes (Nunc, Roskilde, Denmark). After an initial culture period of 2 h at 37°C, 5% CO2, the non-adherent cells were discarded and 10 ml of fresh RPMI supplemented with 10% foetal bovine serum (FBS) (Sigma) (10% FBS/RPMI) added to each tissue culture dish. Cells were then cultured for 5 days at 37°C, 5% CO2, with the addition of a further 10 ml fresh 10% FBS/RPMI after 24 h. Adherent cells were scraped on day 5 and re-plated at 105/well for cytokine analysis and 106/well for RNA extraction in X-Vivo-15 medium (Cambrex, Walkersville, MD, USA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using RNeasy mini kit (Qiagen)
Label
streptavidin-Cy3
Label protocol
standard Illumina protocol
Hybridization protocol
Labelled cRNA was hybridised to Illumina Human-WG6 v3.0 Expression BeadChips (Illumina) for 16 h at 58°C
Scan protocol
Scanned using the Beadarray reader
Data processing
Image data was processed using Genome Studio software. The Sample data were trimmed to include only the genes that were expressed consistently in our monocyte derived macrophages. Only probes that were detected at p < 0.01 in at least 50% of the samples were retained for analysis.
