Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death—either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a cell’s membrane, but become “flipped” to the outer leaflet during the early stages of apoptosis. The second component is the DNA-binding dye molecule PI, which can only enter cells when their membranes are ruptured—a characteristic of both necrosis and late apoptosis.

This video article begins with a review of the concepts behind annexin V and PI staining, and emphasizes how differential patterns of staining can be used to distinguish between cells progressing down different death pathways. We then review a generalized protocol for this technique, followed by a description of how researchers are currently using annexin V and PI staining to better understand cell death.