Differentiation of Human Macrophages from PBMCs

Human macrophages were differentiated from peripheral blood mononuclear cells (PBMCs) as described in Noel et al.^{93 (link)}. Briefly, human blood was obtained from healthy adult volunteers. PBMCs were purified from 10 ml of EDTA-treated human blood using Ficoll-Paque PREMIUM density 1.007 g/ml (GE Healthcare). Contaminating red blood cells were lysed with ACK lysis buffer for 5 min at room temperature (RT). Monocytes were enriched by negative selection using the human Pan Monocyte Isolation Kit (Miltenyibiotec) and LS Columns (Miltenyibiotec). Monocytes were resuspended in RPMI supplemented with 10% FBS (Gemini Bio-products), 55 μM 2-Mercaptoethanol (Gibco), 1 mM Sodium Pyruvate (Gibco), 1x MEM non-essential amino acids (Gibco), and 1x penicillin/streptomycin (Corning). Cells were counted using Trypan blue stain (0.4%; Thermo Fisher Scientific) in a TC20™ Automated cell counter (Bio-Rad) and 2 × 10⁶ monocytes were seeded into six-well plates (Sigma). Human recombinant Macrophage Colony-Stimulating Factor (M-CSF; Biolegend) was added to each well at 50 ηg/ml final concentration on days 0, 2, and 4 after seeding. Medium was changed on day 4. Cells were incubated at 37 °C 5% CO₂ for 6 days to allow differentiation into M0 macrophages.