4.2. Totoxity Evaluation of Alliin on RANKL-Induced Osteoclastogenesis

In order to measure viability, equal numbers of RAW264.7 cells were seeded onto 96-well plates uniformly at 2 × 10³ cells per well, which were maintained in DMEM, supplemented with 10% FBS, and incubated in a humidified atmosphere of 5% CO₂ at 37 °C. When cells had strongly adhered to the plates during the following days, the medium was removed. Fresh medium containing DMEM medium plus 10% FBS, 50 ng/mL RANKL, 50 ng/mL M-CSF, and different concentrations of alliin were added to each well, which were used to induce the osteoclastogenesis fusion and differentiation. Meanwhile, other plates were added only fresh medium containing DMEM medium and different concentrations of alliin. After cells were incubated for 48 or 72 h, the medium was removed and 100 μL Cell Counting Kit-8 (CCK-8) solution was added to each well. After 2-h incubation, the remaining fixed products were analyzed by using multi-detection microplate reader (BioTek Instruments, Winooski, VT, USA) at wavelength of 450 nm. According to the related protocol, the procedures were operated in turn. The wells only containing the CCK-8 reagent were used as blank controls.