Macrophage Differentiation from Monocytes

Mononuclear cells were isolated by Ficoll Hypaque (GE Healthcare) gradient density centrifugation. Monocytes were extracted from blood mononuclear cells by positive selection using magnetic microbeads coupled to anti-CD14 antibodies (Miltenyi Biotec) (34 (link)–36 (link)). Purity assessed by flow cytometry was >95%. Viability determined by trypan blue exclusion was >95%. Monocytes were cultured in RPMI medium 1640 supplemented with 10% (vol/vol) FCS (GE Healthcare) and GM-CSF (50 ng/ml) (Peprotech) or M-CSF (50 ng/ml) (Peprotech) for 1 week to induce M1 or M2 macrophages, respectively. Ultrapure E. coli O111:B4 LPS was purchased from List Biological Laboratories. Polyclonal and monoclonal antibodies (pAbs and mAbs) used for flow cytometry, Western blotting and cytometry by time of flight (CyTOF) are described in Table S1 in Supplementary Material. Unless specified otherwise, all other reagents were obtained from Sigma-Aldrich.