CO reduces osteoclastogenesis by RANKL-treated RAW 264.7 cells in vitro. (A) RAW 264.7 cells treated with 10, 15, and 20 ng RANKL/mL and examined by TRAP staining after 96 h. (B) Upper panel, RAW 264.7 cells exposed to air or 250 ppm CO for 96 h and then stained with 0.05% methylene blue, as described in Methods. The data are the mean from three experiments with triplicate samples; bars, +SD. Lower panel, Western blot analysis of activated caspase 3 expression by RAW 264.7 cells. (C) TRAP staining of RAW 264.7 cells treated with 20 ng RANKL/mL and then exposed to 250 ppm CO. (D) Bar chart showing the inhibitory effect of CO (250 ppm) on RAW 264.7 cells treated as in (C).