Flow cytometry is an effective tool for detecting apoptosis, helping researchers distinguish between live cells, apoptotic cells, and necrotic cells. This guide describes how to use flow cytometry with Annexin V and propidium iodide (PI) staining to detect apoptosis. 1. Sample preparation Collect cells from culture and wash with cold PBS to remove any residual culture medium. Resuspend cells in binding buffer (provided in most apoptosis detection kits) to maintain viability during staining. For best results, perform apoptosis detection soon after treatment or exposure to stressors, as cells may go through apoptotic stages. 2. Annexin V and propidium iodide staining Annexin V conjugated to a fluorophore (such as FITC or APC) is added to detect phosphatidylserine on the outer membrane of cells, which is exposed early in apoptosis. Propidium iodide (PI) is added to distinguish between late apoptotic and necrotic cells. PI only enters cells with damaged membranes, staining DNA in late apoptotic or necrotic cells. Incubate cells with both stains for 15 minutes at room temperature in the dark, as light exposure may degrade the fluorophores. 3. Flow cytometry settings Analyze samples on a flow cytometer, using filters appropriate for Annexin V and PI. Typically, a dual laser setting (488 nm for PI and 633 nm for Annexin V-APC) works well. Make sure forward and side scatter settings are optimized to cover all cell sizes and types. 4. Data analysis Analyze cells using flow cytometry software, dividing them into four quadrants: Annexin V-/PI-: live cells Annexin V+/PI-: early apoptotic cells Annexin V+/PI+: late apoptotic or secondary necrotic cells Annexin V-/PI+: necrotic cells This allows for clear distinction between apoptotic stages. Flow cytometry provides a quantitative, reliable method for detecting apoptosis, providing valuable insights into cellular responses to treatment or stress. Reference [1] Lanzhou Jiang et al, Scientific Reports 2017 (https://lnkd.in/evKCDxZX)