Minodronate and alendronate inhibited osteoclast formation in C7 cells. (A, B) Determination of the appropriate concentrations of minodronate (A) and alendronate (B) that are not cytotoxic to C7 cells. Cells (5000 cells/well) were incubated in 96-well plates for 24 h and then treated with various concentrations of minodronate and alendronate. After 12 days, cell viability was quantified by conducting WST-8 assays. The results are representative of 5 independent experiments. *P < 0.01 compared to the controls. (C-F) Inhibition of osteoclast formation by minodronate and alendronate. C7 cells were cultured for 12 days and then treated with 0.1, 0.25, or 0.5 μM minodronate (C, E) or 0.5, 1, or 2 μM alendronate (D, F). Cells were cultured in the presence of 25 ng/mL RANKL plus 50 ng/mL M-CSF. Cultures were fed every 3 days by replacing with 500 μL of fresh medium with or without minodronate, alendronate, RANKL, and M-CSF. Cultures were fixed and stained for TRAP-positive multinucleated cells (C, D), and TRAP-positive cells (E, F) per well was counted. These results are representative of 5 independent experiments. *P < 0.01 compared to 25 ng/mL RANKL plus 50 ng/mL M-CSF administration. (G, H) Inhibitory effect of minodronate and alendronate on RANKL and M-CSF-induced CTR and cathepsin K mRNA expression. C7 cells were treated with minodronate (G) or alendronate (H) with 25 ng/mL RANKL plus 50 ng/mL M-CSF for 12 days. Total RNA was extracted and the levels of CTR and cathepsin K mRNA expression were determined by real-time PCR. The results are expressed as the ratio of treated to control samples after normalization to GAPDH mRNA levels. The results are representative of 4 independent experiments. *P < 0.01 compared to 25 ng/mL RANKL plus 50 ng/mL M-CSF administration.