MTT Assay for Cell Viability

Cell growth was evaluated using the MTT assay, as outlined in prior works [14 (link),71 (link),72 (link),73 (link)]. Briefly, the Ca9-22 cells were exposed to Rapamycin, whether alone or in combination with different concentrations of Cisplatin (1 to 100 µM), for 24 h. The medium was exchanged with a fresh solution containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT agent (Sigma, M-2128) before incubating the cells at 37 °C. Following three hours in the dark, the purple formazan product generated by metabolically active cells was dissolved with isopropanol 0.4% HCl. The optical density was recorded after 15 min at 550 nm using an iMark reader (Bio-Rad, Mississauga, Ontario, Canada). IC50 was defined as the half maximal inhibitory concentration required to inhibit proliferation.