Osteoclastogenesis assay

Freshly isolated bone marrow macrophages (BMMs) from C57BL/6 mice were plated in T75 flasks and cultured in α-MEM supplemented with M-CSF (50 ng/ml). When cells were confluent, BMMs were seeded in 96-well plates at the concentration of 6 × 10³ cells per well. After overnight incubation to allow attachment, cells were differentiated with RANKL (50 ng/ml) and incubated with different concentrations of fangchinoline. Medium was changed every two days for 5 days or until mature osteoclasts were formed. For investigating which stage of osteoclastogenesis is mostly affected by fangchinoline, BMMs were treated with RANKL (50 ng/ml) for 5 days, while 1μM fangchinoline was added to BMM at either day 1, 3 or 5. Then, the cells were fixed in paraformaldehyde for 10 min, followed by three washes with 1 × PBS. After that, the cells were stained with TRAcP staining buffer for counting multinucleated cells and image acquired using a light microscope.