Cell viability

The MC3T3-E1 cells were seeded in a 24-well plate at a density of 2 × 10⁴ cells/well. After 48 h, the cells were preincubated for 1 h with different concentrations of luteolin before treatment with MG for 48 h. Then cell viability was assessed by MTT assay. MTT assay is particularly useful for this type of analysis. Tetrazolium salts are cleaved to formazan by the succinate-tetrazolium reductase system which belongs to the respiratory chain of the mitochondria, and is only active in metabolically intact cells. Briefly, 20 μl of MTT in Dulbecco’s phosphate-buffered saline (DPBS), were added to each well, and incubated for 2 h. After removal of the solution in the well, dimethyl sulfoxide (DMSO) was added to dissolve formazan products, and the plates were shaken for 5 min. The absorbance of each well was recorded on a microplate spectrophotometer (Zenyth 3100 multimode detector, Anthos Labtec Instruments, Wals-Siezenheim, Austria) at 570 nm.