2.2. MTT Cell Viability Assay

HeLa cells were seeded into a 96-well culture plate at a density of 500 cells/well and allowed to grow in DMEM supplemented with 10% FBS for 24 h before treatments. Thereafter, cells in three reduplicative wells were incubated with increasing concentrations (50, 100, 200, 300, 400, and 500 μg/mL) of flavonoids in A. conyzoides (purchased from the herbal garden of Zhangzhou Health Vocational College, Fujian, China) for 16 h. Cells incubated with sterile ddH₂O were considered the control. For the MTT assay, medium in each well was carefully replaced by 150 μL fresh DMEM+10% FBS with diluted MTT (0.2 mg/mL, Amresco, USA) and incubated for 4 h at 37°C. Afterwards, the incubation medium was removed and formazan crystals were dissolved in 150 μL solution of DMSO in each well. The OD value of each well was quantified by recording the light absorbance at 630 nm using a microplate reader (Bio-Rad, Hercules, USA). The calculation equation of cell survival rate is as follows: cell survival rate (%) = OD_flavonoids/OD_control × 100%.