Isolation of primary human monocyte-derived macrophages.

To isolate peripheral blood mononuclear cells (PBMCs), whole blood was collected from healthy human donors and separated by Ficoll-Paque (Thermo Fisher) gradients. To differentiate cells into macrophages, isolated monocytes were maintained in 100-mm dishes in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% fetal bovine serum [FBS], 2 mM l-glutamine, 100 U/ml of penicillin, and 100 U/ml of streptomycin) supplemented with either human granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 ng/ml), human M-CSF (50 ng/ml), or human GM-CSF (50 ng/ml) plus human interleukin 4 (IL-4; 20 ng/ml). The culture medium was changed every 3 days. Macrophages were considered fully differentiated by 6 to 7 days of culture as defined by a change in morphology. After 6 to 7 days of differentiation, MDMs were activated toward particular phenotypes using different cytokine combinations. For the M1 phenotype, GM-CSF-induced MDMs were stimulated for 18 h with tumor necrosis factor alpha (TNF-α; 20 ng/ml) plus human gamma interferon (IFN-γ; 20 ng/ml). For the M2 phenotype, M-CSF-induced MDMs were activated for 18 h with human IL-4 (10 ng/ml) and human IL-13 (10 ng/ml). For DCs, monocytes were simply maintained for 6 to 7 days in human GM-CSF and human IL-4.