MTT Proliferation Assay

The MTT reagent (Amresco, USA) can be metabolized by active cells to formazan, an insoluble purple dye that can be measured in a spectrophotometer, and was used according to the manufacturer's instructions. Cells were seeded in quadruplicates in 96-well plates (1,600 per well). After 24-h adhesion time, the cells were treated with the compounds chosen, and anticancer product norcantharidin (NCTD) (Sigma, USA) in positive group (Zimu and Zhenxiao, 2015). 0, 24, 48, and 72 h later, 100 μl of MTT reagent were added to each well and the cells were again incubated for 4 h at 37°C and 5% CO₂. After incubation and 10 minutes shaking, the absorbance was measured on a Genios Microplate Reader (Spectra Max190). All experiments were performed in triplicates.