Primary cultures of mouse peritoneal macrophages
Peritoneal macrophages were recovered as previously described (Pepe et al., 2017). Briefly, 5 ml of pre-chilled 0.9% NaCl were injected in the peritoneal cavity using a 21G needle, recovered and centrifuged at 1500 rpm for 8 minutes; cells were incubated with ACK solution (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA; pH 7.3) for 5 minutes at 4°C and seeded at the concentration of 1 x 106 cells/well in 12-wells plate with RPMI (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% endotoxin-free FBS, 1% penicillin/streptomycin and 1% Na pyruvate. After 45 minutes, cells were intensively washed with PBS and incubated in RPMI w/o phenol red with 10% dextran coated charcoal-FBS. Cell numbers were analyzed by counting viable cells after harvesting with StemPro Accutase (Thermo Fisher Scientific) and staining with Trypan Blue (Sigma-Aldrich, St. Louis, Missouri, USA).