Apoptosis Analysis by Flow Cytometry

Apoptosis was analyzed by cytometric analysis, using FITC Annexin V Apoptosis Detection Kit (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, cells were trypsinized, washed twice with cold PBS, and 10⁶ cell/mL were resuspended in 1X binding annexin V-FITC (0.25 µg/mL) and propidium iodide (PI) (1 µg/mL) were added to cell suspension and incubated, protected from light, for 10 min at room temperature. Samples were analyzed using Guava easyCyteflow cytometer (Merck Millipore, Darmstadt, Germany). For each sample, 5000 events were acquired [44 (link)]. Annexin V-FITC is detected as a green fluorescence and propidium iodide is detected as a red fluorescence. Early apoptosis is defined by annexin V+/PI− staining, late apoptosis is defined by annexin V+/PI+ staining, and necrosis is defined by annexin V−/PI+ staining).