Rhodamine conjugated phalloidin solution

BM cells were obtained from the femur and tibia of 6-wk-old C57BL/6 mice, and BMDMs were cultured in α-MEM (Gibco) containing 10% FBS (Sigma-Aldrich) in the presence of 100 ng/ml M-CSF (R&D Systems) for 2 d. Osteoclasts were generated by stimulating BMDMs with 10 ng/ml M-CSF and 100 ng/ml RANKL (Wako Pure Chemical Industries, Ltd.) for an additional 4–5 d or by the co-culture system established by Takahashi et al. (1988) (link). The survival assay was performed as follows. After osteoclasts were generated, both RANKL and M-CSF were removed from the culture (time 0), and osteoclasts were cultured for the indicated times. The survival rate of the cells was estimated as the percentage of morphologically intact TRAP⁺ multinucleated cells compared with those at time 0. Actin ring formation was examined using rhodamine-phalloidin staining. In brief, cells were incubated for 30 min with rhodamine-conjugated phalloidin solution (Molecular Probes). The actin rings formed by osteoclasts were detected with a BZ-8100 fluorescence microscope (KEYENCE). The osteoclast bone resorption assays were performed as previously reported (Miyazaki et al., 2000 (link)). In brief, the cells were cultured on dentine slices for 24 h, and the resorption areas were visualized by staining with 1% toluidine blue and then measured using an image analysis system (MicroAnalyzer).