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Osteoclast differentiation and TRAP staining
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As reported previously16, BMMs were seeded into 96-well plates (1 × 104 cells/well). After 24 h, the cells were cultured in α‐MEM (10% FBS, 30 ng/ml M‐CSF and 50 ng/mL RANKL) with isorhamnetin 3-O-neohesperidoside at a concentration gradient (0, 1, 5, 25 and 50 μM). The medium was changed every 2 days. After fixation with 4% paraformaldehyde, TRAP staining solution was applied to the cells. TRAP-positive cells with more than three nuclei were counted as osteoclasts, which were analysed using Image J software.
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