Gene expression in rheumatoid arthritis synovial macrophages

Macrophages from synovial fluids of five RA patients were isolated by ficoll density gradient centrifugation, followed by positive selection of CD14+ cells using magnetic beads. The patients’ diagnoses were determined by their physicians, in each case a Board-certified rheumatologist at the Hospital for Special Surgery, and were definite RA according to American College of Rheumatology criteria. De-identified samples were processed in an anonymous manner using a protocol approved by the Institutional Review Board of the Hospital for Special Surgery.Human peripheral blood mononuclear cells were isolated from venous blood of independent healthy donors by centrifugation on a Ficoll density gradient. Monocytes were obtained from PBMCs using anti-CD14 magnetic beads, and were >96% pure as verified by flow cytometry. Monocytes were differentiated into macrophages by culturing in RPMI 1640 medium supplemented with 10% fetal bovine serum and M-CSF (20 ng/ml). The viability and purity of macrophages was comparable in all conditionsGene expression was analyzed using Affymetrix microarrays and protocols.