Osteoclast Differentiation from Murine Bone Marrow

Bone marrow cells extracted from femurs and tibiae of 10–12-wk old mice were cultured in α-MEM containing 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin with 20 ng/ml recombinant mouse M-CSF (416-ML; R&D Systems) in plastic petri dishes. Cells were incubated at 37°C in 95% air/5% CO₂ for 4 d and then lifted with 5 mM EDTA in PBS. Recovered BMDMs were cultured in α-MEM containing 10% FBS supplemented with 20 ng/ml mouse M-CSF and 30 ng/ml mouse RANKL (462-TEC; R&D Systems) for 5 d in tissue-culture dishes to induce osteoclast formation (Tang et al., 2009 (link); Zhu et al., 2020 (link)). Mature osteoclasts were characterized by staining for TRAP activity using an Acid Phosphatase Leukocyte Kit (387A; Sigma-Aldrich) and TRAP-positive MNCs (>3 nuclei/cell) counted.