Early detection of apoptosis using a fluorescent conjugate of annexin V

Abstract

Apoptosis of mammalian cells is accompanied by various morphological changes including nuclear condensation, DNA fragmentation and cell surface changes. Methods developed over the past few years have focused on detection of DNA-associated changes that occur rather late in apoptosis. However, detection of apoptosis at early stages, before gross morphological changes, is critical for understanding the pathways of programmed cell death. In this report, we describe a rapid and reliable assay for detecting early stages of apoptosis. This assay is based on the observation that soon after initiating apoptosis, most mammalian cell types translocate phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be specifically detected by staining with fluorescein isothiocyanate (FITC)-labeled annexin V (annexin V-FITC), a protein with a strong, natural affinity for PS. Using this assay, we have detected apoptotic cells in culture, in real time, using fluorescence microscopy and flow cytometry. In combination with vital dye staining, the progressive stages of apoptosis were observed. PS redistribution occurs earlier than DNA-associated changes and membrane leakage. In addition, PS externalization occurs during apoptosis induced by a variety of stimuli. Therefore, the annexin V binding assay provides an excellent indicator for the early stages of apoptosis.