Osteoclast differentiation, TRAP staining, F-actin band staining, and pit formation assay

RAW264.7 cells were seeded into 48-well plates and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin. Cells were subjected to treatments according to experimental requirements. Cells were then stimulated with RANKL (20 ng/ml) for 7 days. Culture medium was replaced with fresh medium every other day. After 7 days, TRAP staining was used to evaluate osteoclast differentiation. Cells were fixed and subjected to TRAP staining. Briefly, cells were submerged in a mixture of 3.0 mg naphthol AS-BI phosphate, 18 mg red violet LB salt, and 100 mML (+) tartaric acid (0.36 g) diluted in 30 ml of 0.1 M sodium acetate buffer (pH 5.0) for 15 min at 37 °C. Multinucleated TRAP-positive cells with at least three nuclei were scored as osteoclasts. RAW264.7 cells were seeded into 48-well plates and cultured for 7 days as previously described. Cells were fixed and stained for F-actin band staining. Briefly, cells were submerged in 5 μl/ml phalloidin (Yeasen, China) for 30 min after treatment with 0.5% Triton X-100 for 5 min. The pit formation assay was performed using Corning osteo assay surface multiple well plates (Corning, USA). RAW264.7 cells were seeded into 96-well plates and cultured for 10 days as previously described. Next, plates were stained with Von Kossa to increase the contrast between pits and surface coating and observed under a light microscope.