Isolation and Cultivation of Murine Bone Marrow Macrophages

Isolation of murine bone marrow cells and culture of bone marrow-derived macrophages were conducted as previously described (Toda et al. 2021 (link)). Briefly, C57BL/6J male mice were euthanized, and femurs and tibias were separated to flush bone marrow cells in precooled PBS. The isolated cells were incubated in erythrocyte lysis buffer and then washed with PBS. After centrifugation at 200 g for 5 minutes at 4°C, the cells were resuspended in RMPI medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin, and 50 ng/ml recombinant macrophage colony-stimulating factor (M-CSF, PeproTech, USA) and incubated in a cell incubator with 5% CO₂ at 37℃. After 7 days, mouse bone marrow-derived macrophages were harvested and stimulated with murine TGF-β1 (1 ng/ml or 5 ng/ml, PeproTech, USA) and bortezomib (5 nM).