Fluorescein isothiocyanate conjugated annexin 5 apoptosis detection kit 1

Cells were harvested and washed twice with ice-cold PBS, after which they were resuspended in 1X binding buffer [0.01 M HEPES/NaOH (pH 7.4), 0.14 M NaCl, 2.5 mM CaCl₂] at a concentration of 10⁶ cells/ml. Subsequently, 100 µl solution was transferred to a 5 ml cell culture tube and was treated with fluorescein isothiocyanate-conjugated Annexin V apoptosis detection kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. The cells were analyzed using flow cytometry (DiVa 8.0.1; BD Biosciences) and a total of 10,000 cells/sample were analyzed to determine the percentage of apoptotic cells.

The cell cycle distribution and apoptotic rate were detected by flow cytometry (BD FACSCanto, United States) and analyzed using ModFitLT software version 5.0. AGS cells were incubated in 6-well plates and treated with resveratrol at different concentrations (0, 12.5, 25, and 50 μM.) after adherent growth. After treatment for 24 h, cells were collected and then fixed with precooled 70% ethanol at 4 °C for at least 2 h. The cell cycle assay was performed with a DNA Content Quantitation Assay Kit (Solarbio, China) according to the manufacturer’s protocol. After treatment, cells were digested and collected with 0.25% trypsin digestion solutions without thylenediamine tetraacetic acid (EDTA), and cell apoptosis was detected using the fluorescein isothiocyanate-conjugated Annexin V Apoptosis Detection Kit I (BD Biosciences, United States). Finally, the samples were transferred to a flow cytometer and measured.