Annexin 5 fluorescein isothiocyanate fitc pi staining kit

Apoptosis was measured by flow cytometry using an annexin V–fluorescein isothiocyanate (FITC)/PI staining kit (BD Biosciences, San Jose, CA, USA). The cells (1.0 × 10⁶/10 mL/dish) were collected, washed with PBS, and resuspended in 200 μL of 1× binding buffer containing annexin V (1:50 according to the manufacturer’s instructions) and 20 ng/sample PI for 15 min at 21–23 °C in the dark. Viable, apoptotic, and necrotic cell counts were quantified using flow cytometry (Becton Dickinson; Franklin Lakes, NJ, USA) and analyzed using CellQuest software. The cells were excited at 488 nm, and the emissions of annexin V at 525 nm and PI were collected using 610 nm band-pass filters. A total of 10,000 cells were analyzed for each sample. Apoptotic cells were expressed as a percentage of total cells ([apoptotic cell count]/[total observed cell count] × 100). Apoptotic cells included annexin V⁺/PI⁻ cells (early apoptosis) and annexin V⁺/PI⁺ cells (late apoptosis).