Apoptosis Assay for the Evaluation of Cisplatin-Induced Apoptosis

GC cell lines were exposed to different concentrations of cisplatin (0–40 µM) (Accord Healthcare Limited, Harrow, United Kingdom) for 48 h to assess their sensitivity. Similarly, to evaluate the effect of TRPV2 blockade by tranilast (Tocris Bioscience, Bristol, United Kingdom) on cisplatin resistance of KATO-III cells, cisplatin (10 µM) was added to cell culture after 10 min since the addition of TRPV2 inhibitor (250 µM). After 48 h of culture, KATO-III cells were recovered by trypsinization and subjected to flow cytometry. In brief, cells were washed twice with cold PBS and then, after centrifugation, resuspended in 100 µL of 1x binding buffer at a concentration of 1 × 10⁶ cells/ml. Subsequently, 5 µL of FITC Annexin V and 5 µL propidium iodide (PI) (BD Biosciences, San Jose, CA, United States) were added, and cells were incubated for 15 min at room temperature (RT) in the dark. For each tube, an adequate volume of 1x binding buffer was added. All samples were acquired, within 1 h, by using a NAVIOS flow cytometer and analyzed by Kaluza software (Beckman Coulter Diagnostics, Brea, CA, United States). A total of 10⁴ events were acquired for each sample. Data from treated samples were normalized as fold change of their untreated controls and were reported as mean ± SE of at least three independent experiments.