Annexin 5 fluorescein isothiocyanate fitc apoptosis assay kit

Cells were routinely transfected and cultured for 48 h and were then digested with EDTA-free trypsin. An Annexin V-fluorescein isothiocyanate (FITC) apoptosis assay kit (BD Biosciences) was used to estimate the apoptosis rate according to the manufacturer's instructions. Cells were suspended in 1X Annexin binding buffer and 5 µl of Annexin V reagent and 3 µl of propidium iodide (PI) reagent were then added to 100 µl of the cell suspension and mixed. The mixture was incubated in the dark for 30 min at room temperature and 400 µl of 1X Annexin binding buffer was then added to each sample to terminate staining.
For cell cycle analysis, a PI/RNase staining kit (BD Biosciences) was used according to the manufacturer's instructions. After treatment, tongue cancer cells were harvested, washed with ice-cold PBS and fixed with 70% ethanol for 24 h at 4°C. After staining with PI in the dark for 30 min at 4°C, the apoptosis rate (the percentage of late apoptotic cells) and cell cycle distribution were evaluated using a FACSVerse flow cytometer (BD Biosciences).

The Annexin V/fluorescein isothiocyanate (FITC) apoptosis assay kit (BD Biosciences, USA) was used to evaluate cell apoptosis. Briefly, cells were suspended in 1 × binding buffer, and cell number was adjusted to 1 × 10⁷ cells/mL. Cells were incubated with annexin V‐FITC and propidium iodide (PI) for 15 min in the dark. The apoptosis rate was then analyzed using a FACSCalibur flow cytometer (BD Biosciences).²⁷