Suppressive effects of COX-2 inhibitor, celecoxib, on RANKL-induced osteoclastogenesis in BMMs. (A) Demonstration of TRAP⁺ cells differentiating from BMMs unstimulated (i) or stimulated (ii) with RANKL. On day 5 with RANKL stimulation, the cells were fixed and stained for TRAP. TRAP⁺ cells appear as red cells. (iii-viii) Dose-dependent effects of celecoxib on the formation of TRAP⁺ multinucleated osteoclasts (MNCs) with RANKL stimulation. BMMs were pretreated (iii, 100 ng/mL; iv, 50 ng/mL; v, 25 ng/mL) with different concentrations of celecoxib or treated (vi, 100 ng/mL; vii, 50 ng/mL; viii, 25 ng/mL) 2 days after RANKL stimulation. On day 5 with RANKL stimulation, the cells were fixed and stained for TRAP (original magnification, × 100). (B) Total numbers of TRAP⁺ MNCs are shown. Note that formation of TRAP⁺ MNCs was inhibited by pretreatment of celecoxib in a dose-dependent manner. The open and filled arrowheads at bottom indicate pretreatment and posttreatment with celecoxib, respectively. Similar results were obtained in 2 additional experiments. Data are expressed as means ± SDs of triplicates.