RANKL-induced ROS production in RAW264.7 cells and BMM cells. (A) RAW264.7 cells and BMM cells were preloaded with DCFH-DA and treated with RANKL (50 ng/mL) for 10 minutes. DCF fluorescence was measured with a confocal laser-scanning microscope as described in “Assay of intracellular ROSs.” Representative microscopic fields are shown. (B) A soluble form of RANK-Fc inhibits ROS production. BMM cells were pretreated for 60 minutes with 10 μg/mL human immunoglobulin G (IgG) or 5 μg/mL RANK-Fc, then stimulated for 10 minutes with RANKL (50 ng/mL). The production of ROSs was assayed as in panel A. Data are expressed relative to the value for nonstimulated cells. (C-D) BMM cells were stimulated with increasing concentrations of RANKL for 10 minutes (C) or with 50 ng/mL RANKL for different time intervals (D), and the production of ROSs was assayed as in panel A. Results are representative of at least 3 independent sets of similar experiments (A-D). Data are expressed as means ± SDs of triplicates.