For the experiments, lipopolysaccharide (LPS, Sigma) was used at a final concentration of 10μg/mL.
Growth protocol
Primary monocytes were extracted from the blood samples of anonymous healthy male donors, donated by the luxembourgish Red Cross. The components of the blood (peripheral blood mononuclear cells - PBMCs -, plasma and erythrocytes) were separated by Ficoll density gradient separation. After a 10 minute centrifugation (1000 g at room temperature without break), the PBMCs layer was collected and the CD14+ monocytes were isolated by using the MACS® technology (magnetic separation) from Miltenyi Biotec (Bergisch Gladbach, Germany). Isolated CD14+ monocytes were plated at 4x106 cells per well in 6-well plates and differentiated for 11 days into macrophages using RPMI 1640 medium (VWR, Radnor, Pennsylvania) supplemented with 10% human serum, off the clot, type AB (A&E Scientific, PAA, Pasching, Austria, lot number: C02108-1021), 0.1mg/mL streptomycin, 100U/mL penicillin and 0.1mM L-glutamine (Invitrogen, Life Technologies, Carlsbad, California). During the differentiation, the medium was replaced with fresh medium on day 4 and day 7.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by using TRIZOL reagent (Sigma-Aldrich)
Label
biotin
Label protocol
The Ambion® WT Expression Kit For Affymetrix® GeneChip® Whole Transcript (WT) Expression Arrays Part Number 4425209 Rev. C 09/2009
Hybridization protocol
GeneChip® WT Terminal Labeling and Hybridization User Manual for use with the Ambion® WT Expression Kit P/N 702808 Rev. 6
Scan protocol
Affymetrix® GeneChip® Command Console® User Manual (P/N 702569).
Data processing
The data were preprocessed, normalised and analysed using the GCRMA and limma packages from R/Bioconductor software environment. The CDF was used according to GPL15034 and submitted the unique list of IDs and their expression values.
