4.5. TRAP Staining

For TRAP staining osteoclast cultures, cells were washed with PBS, fixed with 4% formaldehyde in PBS for 10 minutes then incubated with prewarmed TRAP staining solution (50 mM sodium acetate buffer pH 5.0, 0.1% Triton X-100, 30 mM sodium tartrate, 100 µg/mL napthol AS-MX, 3 µg/mL fast red violet LB) at 37 °C for 5–10 minutes until stained sufficiently. Color development was stopped by washing twice with PBS. Cells were then counterstained with DAPI. The experiments shown in Figure 4A–B were performed independently three times with similar results. Each experiment consisted of four wells per group. For quantitation, three random fields were photographed for DAPI and TRAP from each of the four wells using an Olympus BX-51 microscope equipped with Olympus DP71 digital camera, and the number of DAPI-stained nuclei and TRAP-positive cells determined using NIH ImageJ. TRAP-positive cells containing three or more nuclei were classified as osteoclasts.