Annexin V is a Ca2 -dependent phospholipid-binding protein that specifically binds to phosphatidylserine (PS) Normally PS is localized on the inner leaflet of the cell membrane lipid bilayer During the early stages of apoptosis PS translocates from the inner leaflet to the outer surface of the cell membrane exposing it to the extracellular environment Due to Annexin V s high affinity for PS fluorescently labeled Annexin V (HF488) can be used as a fluorescent probe to detect apoptotic cells through flow cytometry or fluorescence microscopy with staining taking only 10-20 minutes 7-AAD can specifically bind to double-stranded DNA resulting in red fluorescence Normally 7-AAD cannot pass through intact cell membranes however it can penetrate the cell membranes of late-stage apoptotic and dead cells staining their nuclei Combined staining with Annexin V-HF488 and 7-AAD allows differentiation of cells in various stages of apoptosis and dead cells