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On day 4, cells were gently washed twice with pre-warmed PBS (phosphate buffered saline), fixed with 4% paraformaldehyde (PFA) and stained for tartrate-resistant acid phosphatase (TRACP), which is considered a chemical marker of osteoclasts . Briefly, the fixed cells were incubated in a solution of naphthol AS-BI phosphate and fastred TR salt (Sigma) in 0.2 M acetate buffer (pH 5.2) containing 100 mM sodium tartrate (Sigma) for 30 min at 37°C. TRACP-stained cells were washed three times with PBS, counterstained with DAPI (diamidino-2-phenylindole, 0.165 μmol in PBS containing 0.1% TritonX-100) for 10 min and viewed with a Nikon Elipse E1000 microscope.TRACP+ multinucleated cells containing three and more nuclei were categorized as osteoclasts. The number of TRACP-positive osteoclasts and the number of nuclei within these osteoclasts were counted in 5 random fields of view (FOVs), respectively.A fusion index (FI, #nuclei in the fused cells/total nuclei × 100) was calculated for these cell lines.
