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.
Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes. Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanism leading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases (also termed caspases) have been discovered and implicated as the common effectors of apoptosis. In this report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectrum inhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events were followed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetic tetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, such as morphological characteristics and DNA fragmentation. These results suggest that staurosporine-induced apoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.