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 (HF647) can be used as a probe in combination with flow cytometry or fluorescence microscopy for detecting apoptosis and the staining process requires only 10-20 minutes 7-AAD specifically binds double-stranded DNA emitting red fluorescence Normally 7-AAD cannot penetrate intact cell membranes However in late-apoptotic and dead cells membrane integrity is compromised allowing 7-AAD to enter and stain nuclear DNA Co-staining with Annexin V-HF647 and 7-AAD thus differentiates cells in various apoptotic stages and dead cells