Osteoclastogenesis from Bone Marrow Macrophages

BMMs were obtained as described previously [66 (link)]. Briefly, whole bone marrow cells were cultured with 10% FBS, 1% PSG, 10 ng/mL of M-CSF (R&D Systems, Minneapolis, MN, USA) for 12 h. Non-adherent bone marrow cells were then replated in Petri dishes with 10 ng/mL M-CSF and adherent BMMs were harvested as osteoclast precursors 4–5 days later. To generate pre-osteoclasts or mature osteoclasts, BMMs were cultured with 30 ng/mL M-CSF and 30 ng/mL RANKL (R&D Systems) for 3 or 5 days at 37 °C. To enumerate osteoclasts, the cells were stained with tartrate-resistant acid phosphatase (TRAP) using a Leukocyte Acid Phosphatase Assay Kit following the manufacturer’s instructions (Sigma-Aldrich). A giant osteoclast formation was identified as a multinucleated (>10 nuclei) TRAP-positive cell.