Biotek system

Briefly, approximately 1.5 × 10³ RKO cells were seeded into 96-well plates per well with 5 repetitions in each group. After the cells adhered, 10 μL of CCK-8 reagent (Yeasen) was added to 100 uL of cell culture medium and mixed thoroughly; then, the mixed reagent was added to each well of the whole plate. After that, the 96-well plate was and incubated at 37 °C for 2 hours in the dark. The whole plate was then taken into the Biotek system to measure the absorbance value at 450 nm. The cell proliferation curves were plotted as the measured values of each day by deleting the minimum and maximum values.

Six thousand cells per well were seeded in 96-well plate. The cells were then incubated with indicated agents for different times, followed by the addition of 5 mg/mL MTT (Biosharp, China). After incubation for 4 h at 37°C, the supernatants were removed. A volume of 150 μL of DMSO were added, and the absorbance value (optical density) at 490 nm was measured by Bio-TEK system.