Macrophage Differentiation and Polarization

Macrophages was generated as previously described [27 (link)]. Briefly, bone marrow cells were isolated from the femurs and tibias of C57BL/6 mice and then seeded in cell culture dishes and cultured with complete DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 20 ng/mL recombinant mouse M-CSF (PeproTech) at 37°C in a CO₂ incubator for 5 days to differentiate into macrophages. The cells were washed twice with phosphate-buffered saline (PBS) every other day, and fresh medium was added. On day 6, nonadherent cells were removed and adherent cells were cultured with 20 ng/mL mouse recombinant IL-4 (PeproTech) or 1 μg/mL LPS (Sigma) for 24 h, either with or without L-4F (0.25 μg/mL). The differentiated, adherent, live macrophage population was detached from the plate with a solution containing trypsin (Gibco), and the cells were processed for phenotypic characterization. Based on the specific expression of a number of surface markers, including CD11b, F4/80, MHC II and CD206, the cells were sorted using flow cytometry.