A Real-Time Annexin V Method for Monitoring Programmed Cell Death

We developed a homogeneous luminogenic annexin V binding assay to detect the occurrence of apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The annexin-luciferase fragment fusion pairs have only modest affinity for each other, thus luminescence remains low in the presence of viable cells. When annexin V in the fusion proteins binds in close proximity to the phosphatidylserine exposed on the surface of apoptotic cells, the luciferase fragments reconstitute to form an active enzyme and generate luminescence. The reagent is added directly to cells in culture providing a homogeneous protocol that does not require cell washing steps typically needed with fluorescent annexin V binding assays used for flow cytometry. The real time method has been used to monitor the kinetics of apoptosis and secondary necrosis in several model systems by repeatedly recording luminescence and fluorescence from the same samples of cells.