Annexin 5 fluorescein isothiocyanate and propidium iodide apoptosis detection kit

For the cell cycle assay, the PC cells were harvested at exponential growth phase, and single-cell suspensions containing 1 × 10⁵ cells were fixed with 70% alcohol. Then, the cell cycle was monitored using propidium iodide staining and detected with a FACScan flow cytometer (BD Biosciences, USA). For the cell apoptosis assay, early and late apoptotic cells were evaluated by Annexin-V Fluorescein Isothiocyanate and Propidium Iodide Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions. Finally, the stained cells were analysed with a FACScan flow cytometer.

The A172 and U251 cells were obtained from each group, inoculated in six-well plates at 2 × 10⁵ cells/well, and cultured for 48 h. The cells were then rinsed with PBS and digested in trypsin. After digestion was terminated, the cell suspension was transferred to 15-mL centrifuge tubes and centrifuged at 800 rpm for 3 min. The supernatant was removed, and the cells were resuspended in PBS. Centrifugation was repeated, and the supernatant was removed again. This process was repeated twice. The cells from each group were then resuspended in 500 µL PBS and transferred into 1.5-mL Eppendorf (EP) tubes. Cellular apoptosis was measured using an Annexin V-Fluorescein Isothiocyanate and Propidium Iodide Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA). Apoptosis rates were measured using FACSCalibur flow cytometry (BD Biosciences) according to the manufacturer’s instructions. Apoptosis rate = Up Right(UR) + Lower Right(LR).