Osteoclast Differentiation and TRAP Staining Assay

To assess osteoclast differentiation, 1.0×10⁴ BMMs were seeded in triplicates in 96-well plates and cultured in complete α-MEM supplemented with M-CSF (30 ng/ml), RANKL (50 ng/ml), and magnoflorine (0, 50, 100, and 200 μM). The media was changed every other day until multinucleated pancake-shaped osteoclasts were observed (day 5–7). After fixing for 20 min with 4% paraformaldehyde, the cells were incubated in TRAP staining solution for 1 h at 37°C. Multinucleated cells which were positive for TRAP staining and contained more than three nuclei were identified as osteoclasts. Digital images were captured with an optical light microscope (Olympus, Tokyo, Japan), and the numbers of osteoclasts and the areas of cell spread were then quantified using Image J software (NIH, Bethesda, MD, USA).