2.3. Cell Viability Assay for Benomyl and Valerenic Acid

The concentrations of benomyl and VA used in this study were chosen based on the results obtained from the cell viability assay. The effects of benomyl and VA on cell viability were evaluated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The MTT tests were carried out separately for benomyl and VA. According to the process, 5 × 10⁴ cells in 100 µL of growth medium were incubated in 96-well plates for 24 h before treatment. Subsequently, benomyl and VA were administered to the cells separately. After exposure, the cells were incubated with MTT for 3 h, and after incubation, the medium was discarded and 100 uL of DMSO was added to each well. The absorbance was detected using a microplate reader (Biotek, Epoch, Vermont, USA) at 570 nm. For calculation of the IC₅₀ and IC₃₀ values of benomyl through the MTT assay, solutions with concentrations equal to 2.5, 5, 10, 20, 40, and 60 µM were used, whereas concerning VA, the concentrations of the tested solutions were equal to 300, 400, 500, 600, and 700 µM. After calculation of the IC₅₀ values of benomyl and VA, further experiments were carried out to determine the IC₃₀ values. The cells were exposed to 6 µM of benomyl (i.e., B6) for 6 h to achieve an induction of cellular stress. Then, in order to evaluate the effects of VA, 100 μM (VA100) and 200 µM (VA200) of VA were chosen for a 24 h cell exposure following benomyl exposure.