4.4. MTS Cell Viability Assay

Cell viability assay was measured using the Cell Titer 96 aqueous non-radioactive cell proliferation assay (Promega, Madison, WI, USA) according to the method described by Kim and Jang [44] with slight modifications. In brief, HepG2 cells were seeded in a sterile flat-bottomed 96 well plate and incubated at 37 °C for 24 h in a humidified incubator containing 5% CO₂. Different concentrations of extracts (1, 10, 50, and 100 µg/mL) were prepared directly in fresh serum-free MEM media and 100 µL of each treatment was added to each well and incubated for 24 and 48 h. Next, 20 µL of the MTS reagent in combination with the electron coupling agent phenazine methosulfate was added into each well and allowed to react for 1 h at 37 °C. After 2 min of shaking at minimal intensity, the absorbance at 490 nm was measured using the EnSpire PerkinElmer Multimode Plate Reader. Controls (cells with media containing DMSO (≤ 0.1%)) and blanks (wells containing media without cells) were assessed under the same conditions. The cell viability values were determined using the equations below.

Three separate experiments were conducted, each consisting of three distinct readings.