Bone Marrow-Derived Macrophage Isolation and Culture

For the in vitro osteoclastogenesis assay, mice bone marrow-derived macrophages (BMMs) were collected as previously described (Chen et al., 2019b). Briefly, after euthanasia 8-week-old C57BL/6J mice in CO₂ condition box, BMMs were isolated from the mouse tibias and femurs by flushing the bone marrow with α-MEM. The flushed cells were collected and cultured in α-MEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. During the morning of the second day, the non-adherent cells were collected and cultured in Petri dishes with M-CSF (30 ng/ml) to select BMMs. After incubation for 3 days, the adherent cells (BMMs) were detached using a cell-free enzyme and collected for further culture in an induction medium to induce osteoclast differentiation. All animal experiments were approved by IACUC at Chonnam National University (approval number: CNU IACUC-YB-2019-46).