Quantification of Osteoclast Differentiation

To detect the polarization and resorptive activity, we performed the F-actin ring stain as previously described 27 . MBMs were cultured directly on the 24-well plates or the bovine cortical bone slices in the 24-well plates. Then cells were cultured with medium containing M-CSF/RANKL for 5 days. Cells were fixed with 3.7% formaldehyde for 15 min, washed by cold PBS 3 times, and then permeabilized in 0.2% Triton X-100 for 10 min at room temperature. Next, cells were blocked with 1% goat serum and incubated with rhodamine phalloidin (Life Technologies, USA) and primary antibody-mouse-anti-Ctsk (Santa Cruz, sc-48353) overnight. The following day, cells were then rinsed with PBS and incubated with FITC anti-mouse IgG secondary antibody (Thermo Scientific) for 90 min. Lastly, the nuclei were stained with DAPI (1µg/mL, Sigma). The stained cells were visualized under a fluorescence microscope (Leica Texas Red filter) 30 (link). For quantification of IF stain, we used NIH Image J to perform counts.