Generating M1 and M2 Macrophages from PBMCs

PBMCs were prepared from leukopacks (Etablissement Français du Sang) or blood collected in ethylene-diamine-tetraacetic acid (EDTA) tubes from donors and patients after centrifugation through a Ficoll density cushion. Monocytes were isolated from PBMCs by CD14 positive selection using magnetic beads coated with anti-CD14 antibodies (Miltenyi Biotec). CD14⁺ monocytes were differentiated into macrophages by cell culture (Ghigo et al., 2010 (link)). To obtain M1 macrophages, macrophages were stimulated with 20 ng/mL recombinant human IFN-γ (Tebu-bio) for 18 h. M2 macrophages were obtained by incubating macrophages with 10 ng/mL IL-10 or 20 ng/mL IL-4 (R&D Systems) for 18 h.