Figure 2.

C4-2B CM induces osteoclastogenesis in the absence of osteoblast/stromal cells, and OPG inhibits the osteoclastogenesis in vitro. Single-cell suspensions (10⁵ cells/well) of RAW 264.7 cells were plated in a 24-well plate on top of a sterile coverslip in RPMI plus 10% FBS. Cells were grown for 12 hours, then the media was changed to RPMI plus 0.5% FBS. CM from C4-2B cells was harvested (as described in Methods) and added to a final concentration of 25% (vol/vol). Immediately, recombinant human soluble RANKL (10 ng/ml) or the indicated concentration of recombinant mouse OPG or vehicle (1% BSA in PBS) was added. Osteoclasts were identified as TRAP-positive multinucleated (>3 nuclei) cells. (a) Representative pictures of cultures stained for TRAP. (b) Osteoclasts per coverslip were quantified. Samples were evaluated in quadruplicate. Results are reported as mean (±SD). Data were analyzed using one-way ANOVA. ^AP < 0.001 compared with control culture; ^BP < 0.01 compared with the CM-treated group; ^CP < 0.001 compared with the CM-treated group.