Isolation and Culture of Bone Marrow-Derived Macrophages (BMDM)

Wild-type C57BL/6 mice were exploited to isolate BMDM from the bone marrow as previously described (Ying et al., 2013; Kozicky and Sly, 2019). Briefly, the mouse was euthanized and sterilized with 70% ethanol. The skin and fur were cleared off each of the hind legs, the muscles were trimmed, and the tibias and femurs exposed. A 26-gage needle was used to flush the bone marrow into a 50 mL conical tube. After centrifugation at 300 × g at 4°C for 5 min, BMDM were initially acquired and cultured in DMEM supplemented with 10% FBS. Importantly, recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) with a dose of 20 ng/ml were necessary to induce differentiation of BMDM into normal macrophages (M0) over time. On day 7 after initial culture, BMDM were observed under an inverted microscope and then ready for use.