In vitro Osteoclast Differentiation and TRAP Staining

Osteoclast precursors obtained from BMMs were loaded into a 24-well plate at a density of 5 × 10⁴ cells/well and treated with 30 ng/ml RANKL (R&D Systems) in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin and 50 ng/ml M-CSF for 4 days. The culture medium was replaced every 2 days. After osteoclast differentiation, the cells were fixed with 4% paraformaldehyde for 10 min and then stained for TRAP according to the manufacturer’s instructions of Acid Phosphatase Kit (Sigma-Aldrich). The TRAP- positive cells containing three or more nuclei were recorded.