Effect of AS extract on RANKL-induced osteoclast differentiation. (A) BMMs were cultured for 4 days with M-CSF (20 ng/ml) and RANKL (40 ng/ml) in the presence of varying concentrations of AS extract. Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with TRAP solution. (B) TRAP-positive cells were counted as osteoclasts. Asterisk indicates a statistically significant difference (p < 0.05) between control and treated. (C) Cytotoxicity of AS extract on BMMs. BMMs were cultured for 3 days with varying concentrations of AS extract in the presence of M-CSF (20 ng/ml). XTT solution (50 μl) was added to each well after 3 days, and plates were incubated for 4 h. The optical density was measured at 450 nm. (D) RAW 264.7 cells were seeded at 3000 cells/plate in 48-well plates and cultured with the indicated concentrations of AS extract for 4 days. After 4 days, the cells were fixed and stained with Hematoxylin. Colonies with 50 or greater cells were counted. Similar results were obtained in at least 3 independent experiments.