Murine and Human Macrophage Culture

RAW246.7 murine macrophages (ATCC, TIB71) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Corning, United States) supplemented with 10% fetal bovine serum, 1 mM glutamine, 100 U/μL penicillin and 100 μg/mL streptomycin (all from EuroClone, Italy). The cells were grown in tissue culture flasks or multiwell plates, at 37°C and 5% CO₂. Human monocyte-derived macrophages (HMDMs) were differentiated in vitro from monocytes isolated from the buffy coats of healthy donors, as previously described (Del Porto et al., 2011 (link)). Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation (Lympholyte; Cedarlane, Hornby, CA, United States), and were selected with an anti-CD14 monoclonal antibody coupled to magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD14⁺ cells were differentiated for 7 days in Roswell Park Memorial Institute (RPMI) 1640 (Gibco-BRL, Invitrogen Corporation, Carlsbad, CA, United States) supplemented with 20% fetal bovine serum and 100 ng/mL recombinant macrophage colony stimulating factor (PeproTech Inch, Rocky Hill, NY, United States).