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.