The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay measures the real-time exposure of phosphatidylserine (PS) on the outer leaflet of cell membranes during the apoptotic process. Annexin V luciferase fusion proteins supplied in the assay reagent bind to PS during early apoptosis and are detected with a simple luminescence signal. The assay reagent also includes a DNA-binding dye, which enters the cell and generates a fluorescent signal upon loss of membrane integrity. The combination and timing of luminescent (annexin V binding) and fluorescent (DNA release) signals is used to differentiate secondary necrosis occurring during late apoptosis from necrosis caused by other cytotoxic events.