Induction of Murine Macrophage Polarization

Bone marrow was flushed from mouse femurs and tibias and dissociated into a single-cell suspension. After red blood cell depletion, cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin solution. To induce BMDM differentiation, 10 ng/mL macrophage colony-stimulating factor (M-CSF) (416-ML, R&D Systems) or 15% L929-conditioned media were added every 2 days for 7 days [24 (link)]. BMDMs were stimulated with 100 ng/mL LPS (L4391, Sigma) and 20 ng/mL IFN-γ (315-05, PeproTech) to induce M1 polarization, or 10 ng/mL IL-4 (214-14, PeproTech) and 10 ng/mL IL-13 (413-ML, R&D Systems) to induce M2 polarization. For MMe activation, BMDMs or RAW264.7 cells were treated with 0.4 mM palmitate, 10 nM insulin, and 30 mM glucose for 24 h [25 (link)].