Isolation and Expansion of Mouse Bone Marrow-Derived Macrophages

Mouse bone marrow-derived macrophage (BMMØ) isolation and ex vivo expansion was performed as previously described with slight modifications [14 (link)]. Femur and tibial bone marrow cavities were flushed, resuspended in the DMEM complete medium [(DMEM supplemented with 10% (vol/vol) FBS, 1% (vol/vol) streptomycin/penicillin and 50% (vol/vol) and L929 conditioned medium (as a macrophage colony-stimulating factor (M-CSF))] was passed through a 70-μm-cell strainer and then incubated in a humidified chamber at 37 °C with 5% CO₂. After differentiation for 5–6 days, cells were subcultured (passage 1), maintained, and used for flow cytometry analysis using CDD11b-APC (1:100; #553312; BD Biosciences) and F4/80-PE (1:100; #565410; BD Biosciences).