2.3. Cell proliferation assays
Proliferation of EGF, RHC and RHC/EGF were assessed using an MTT assay. NIH/3T3 cells (3×103 cells/well) were seeded into 96-well plates. After 24 h of culture, the medium was changed to maintenance medium (MEM with 0.4% FBS), and the cells underwent starvation for 6 h. The cells were then cultured in maintenance medium containing 0, 0.5, 1, 2, 4, 8 or 16 nmol/L RHC, EGF or RHC/EGF for 48 h. The effect of RHC, EGF or RHC/EGF on NIH/3T3 cells proliferation was detected by the MTT assay according to the manufacturer’s instructions. Briefly, 20 μL of 0.5% MTT solution was added to each well in the dark, and the cells were incubated at 37°C in a 5% CO2atmosphere for 4 h. After removal of the medium, 100 μL of dimethyl sulfoxide was added, and the absorbances at 570 nm were measured in a microplate reader (MK3, Thermo, Waltham, MA, USA). Each assay was performed in triplicate.