Cytotoxicity Evaluation of GSPE Extract

U2OS, A549, and SKOV-3 cells (5000/well) were seeded in a 96-well plate. Next day, treatment with different concentrations of GPSE extract were given for 48 h. DMSO was used as a solvent control of which the volume was matched to the amount used for respective GSPE- and ARC-treated cells in all the experiments. Cytotoxicity assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) as described earlier [23 (link)]. Briefly, viability of control and treated cells was evaluated based on their metabolic activity as determined by the conversion of MTT (yellow) by the mitochondrial NADH dehydrogenases of living cells into formazan (purple). The statistical significance of the results was determined from three independent experiments including triplicate sets in each experiment. For WST assay, cells were treated with different concentrations of GPSE extract for 48 h followed by addition of premix WST-1 (Takara Bio Inc., Shiga, Japan). Cell viability (based on their metabolic activity) was measured at 450 nm with a reference wavelength at 630 nm.