4.4. Cell Viability Assays

MES-SA and MES-SA/Dx5, BMDM, and HUF cell lines were seeded in 96-well plates and incubated for 24 h. Cells were then treated with increasing concentrations of PC and TR for 72 h. MES-SA and MES-SA/Dx5 cells were also treated with increasing concentrations of Dox, cisplatin, 5-fluoro-uracil, and paclitaxel for 72 h. Cell viability was determined through the colorimetric methylthiazol tetrazolium (MTT) assay. This method is based on the ability of mitochondrial succinate deshydrogenases in living cells to reduce a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, leading to the production of blue formazan crystals [63]. Thus, this quantitative colorimetric assay has been widely used to assess the effects of drugs on cell growth [64]. After MTT addition (0.5 mg/mL), the plates were incubated at 37 °C in a 5% CO₂ humidified atmosphere. After a 3 h incubation, the medium was removed, and formazan crystals were dissolved with acidic isopropanol (0.1 N HCl in absolute isopropanol). Absorbance at 570 nm was measured using the Biochrom Asys UVM 340 microplate reader (Biochrom Ltd., Holliston, MA, USA). Results are expressed as a percentage of control values. The inhibitory concentrations at 50% (IC₅₀) for each drug, defined as the concentration of drug that inhibits cell proliferation by 50%, were calculated using a four-parameter nonlinear regression with GraphPad Prism version 6 software (GraphPad Software, San Diego, CA, USA). The resistance index (RI) was calculated by the ratio of the IC₅₀ of resistant cells to that of parental cells.