Human monocyte-derived macrophages polarized by GM-CSF (M1) or M-CSF (M2) in the presence or absence of 5 micromolar methotrexate

Identification of genes differentially expressed between human monocyte-macrophages generated in the presence of either GM-CSF (termed M1) or M-CSF (termed M2) and in the presence (+MTX) or absence (-) of methotrexate Human peripheral blood monocytes from three independent healthy donors (d1, d4 and d7) were isolated by anti-CD14-labeled magnetic microbeads. CD14+ monocytes were exposed to 5 micrmolar methotrexate (MTX) and cultured for 7 days in RPMI 10% FCS containing either GM-CSF (M1) or M-CSF (M2). Total RNA from each condition was extracted and purified using the RNeasy kit (Qiagen). Labelled RNA was used as hybridization probes on human Agilent human whole genome array (G4845A). All experimental procedures were performed following manufacturer instructions. Microarrays were scanned with a GenePix 4000B (Axon Instruments) scanner. Scanned images and raw data were pre-processed with GeneSpring v12 (Agilent).