Osteoclast-like cells were generated in culture from RAW 264.7 cells (transformed murine monocytic cell line), purchased from the American type culture collection (ATCC, VA, USA) (21) . RAW 264.7 cells were cultured to confluence in a 75 cm 2 flask in GIBCO High Glucose 1X Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, CA, USA) containing 4.5 g/L D-Glucose, Lglutamine, and 110 mg/L sodium pyruvate, with the addition of 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin added (10,000 U/mL; Invitrogen, USA). Confluent cells were harvested by scraping, centrifuged at 1,500 RPM, and resuspended in 25 mL of culture medium. The number of cells in the suspension was measured using a Bright-Line Hemocytometer (Hausser Scientific, PA, USA) and seeded at a concentration of 2×10 3 cells/well in 96-well culture plates (Corning) and placed in the CO 2 incubator overnight to allow the cells to attach to the surface. After 24 h, the culture medium was replaced with media containing 25 ng/ml macrophage-colony-stimulating factor (M-CSF) and 50 ng/mL RANKL (PeproTech, NJ, USA). Osteoclasts were successfully generated by dosing with M-CSF and RANKL every 48 h over the course of 7 days. To confirm the generation of multinucleated osteoclast-like cells, the cultured cells were stained for the enzyme tartrate-resistant acid phosphatase (TRAP) using the Leukocyte Acid Phosphatase TRAP Kit from Sigma-Aldrich (St.Louis, MO, USA), according to the manufacturer's instructions. TRAP-positive multinucleated osteoclasts were visualized by light microscopy and photographed.