3.3. In Vitro Anti-Proliferative Assay

The tested cells were plated in 96-well microtiter plates with a density of 2,000 cells/well and incubated in a humidified atmosphere with 5% CO₂ at 37 °C for 24 h. The cells were then exposed to tested compounds of different concentrations in triplicate. After incubated for 96 h, 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (2 mg/mL) was added to each well and incubated for an additional 4 h. Then the suspernatant was discarded and the fromazan was dissolved in 200 μL of dimethyl sulfoxide (DMSO). The plate was oscillated subtly at room temperature for 10 min before the absorbance was measured at 570 nm by a microplate reader. Cell inhibitory ratio was calculated with the following formula:

The GI₅₀ was determined as the concentration that caused half inhibition of cell proliferation. All experiments were performed three times independently, and the results were reported presented as the mean.