2.8.1. MTT cell viability assay

In-vitro cytotoxicity evaluation was performed in human cervical cancer cells (HeLa cells) using MTT (3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide) assay (Al-Qubaisi et al., 2011). Briefly, HeLa cells were seeded in 96 well plates (1 × 10⁴ cells/well) in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum and 1% antibiotic antimycotic solution in a humidified incubator at 37 °C with continuous flow of 5% CO₂. Then cell were treated with different concentrations (50, 250, 500, and 1000 µgmL⁻¹) of test sample and incubated at 37 °C for 24 h. After that, freshly prepared MTT (0.5 mgmL⁻¹) was added and incubated for 4 h at37 °C. After the exposure period, the MTT containing medium was discarded from the cells and washed with 400 µL of phosphate buffer solution (PBS, pH 7.4). After washing, the obtained crystal was dissolved in 400 µL of dimethyl sulfoxide (DMSO) and mixed thoroughly. Microplate reader was used to measure the cell viability at the absorbance of 570 nm. The percentage of viability was calculated by using the following equation: