Cell counting kit-8 (CCK-8) cell proliferation assay

RF/6A cells were inoculated into 96-well plates at 5x10³ cells/well in 100 µl culture medium. Following incubation overnight in a 5% CO₂ incubator at 37˚C, the cells were treated according to their assigned group for 24 h. Subsequently, 10 µl CCK-8 solution was added to each well, followed by incubation for 4 h at 37˚C. This was repeated three times independently of each other. An ELISA plate reader at the wavelength of 450 nM was used to determine the absorbance and the optical density was recorded in order to calculate the cell proliferation rate.