3.4. TRAP Staining and Activity Assays

Multinucleated osteoclasts were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and then stained with the Acid Phosphatase, Leukocyte (TRAP) kit (Sigma-Aldrich, MO, USA) according to the manufacturer’s instructions. TRAP-positive multinucleated cells were visualized under a microscope, and the percentage of osteoclast area was calculated. To measure TRAP activity, cells were washed with physiological saline and lysed with extraction buffer (physiological saline including 1% NP-40), and then incubated with reaction buffer (0.5 M sodium tartrate buffer, pH 5.2, 0.5 M sodium acetate, 12.5 mM pNPP). After 1 h, an equal volume of stop solution buffer (0.5 N NaOH) was added to the reaction. Absorbance was measured at 405 nm after color formation. TRAP activity was presented as the relative ratio of the control.