CCK-8 assay

The proliferation of MC3T3-E1 cells and cytotoxic effect of BDNF or 7,8-DHF on RAW264.7 cells were determined by CCK-8 assay according to the manufacturer’s instructions. Cells were seeded in 96-well plates. BDNF was initially reconstituted in water to 0.1 mg/mL and then diluted in a buffer containing 0.1% bovine serum albumin for extended storage. 7,8-DHF was dissolved in DMSO at 100 mM as a stock solution and diluted with medium. A final DMSO concentration in the culture was less than 0.01% and did not show observably artificial or cytotoxic effects. After 24 hr, the medium was replaced with fresh medium containing different concentrations of BDNF or 7,8-DHF. Cells were cultivated for definite time, followed by CCK-8 solution for 4 hr at 37°C. The absorbance of the resulting solution at 450 nm was measured via a Microplate Reader (Eon, BioTek, Winooski, VT).