Cell morphology of RAW264.7. RAW264.7 cells were cultured in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with various concentrations of LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 6 days, then the cells were fixed and stained with TRAP kit on day 1, 2, 3 and 6. The cell morphology were observed using light microscopy, The number of TRAP-positive staining multinuclear cells increased gradually since 72 h (A), and reached the top amount at day 6 (B). A small number of TRAP-positive cells were observed at day 6 in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with 50 nM LLDT-8 (C). TRAP-positive cells were not observed in the culture medium without RANKL and M-CSF (D). (A-C) 400-fold amplification, bar = 25 μm; (D) 200-fold amplification, bar = 50 μm.