Binding solution

Cells were incubated in 6-well plates at a density of 4 × 10⁵ cells/well with different doses (0, 0.5, 1, 2, 4, 8 mM) of VC at 37°C. After 16 h, the cells were harvested with 0.05% trypsin solution, rinsed twice with PBS and centrifuged at 1500 U/min for 5 min. Next, the cells were stained with fluorescein isothiocyanate-labelled Annexin V (BD Pharmingen, San Jose, CA, United States) and counterstained with propidium iodide (PI; BD Pharmingen, San Jose, CA, United States). Finally, the cells were resuspended in binding solution (BD Pharmingen) and analyzed using a flow cytometer (CytoFLEX S, Beckman Coulter, Fullerton, CA, United States).

Adenocarcinoma cells (5×10⁵/well in 6-well plates in 3 mL) were first incubated with or without pantoprazole (100 μM) for 24 h at 37°C. Then, vitamin C was administered at different concentration. After 16 h, the cells were detached using 0.05% trypsin solution, washed two times with PBS and centrifuged at 1,500 rpm for 5 min. Then, the cells were stained with fluorescein isothiocyanate (FITC)-labelled annexin V (BD Pharmingen, San Diego, CA, USA), counterstained with propidium iodide (PI; BD Pharmingen), resuspended in binding solution (BD Pharmingen), according to the manufacturer’s instructions (BD Pharmingen), and finally analyzed by flow cytometry (CytoFLEX S, Beckman Coulter, Pasadena, CA, USA).