A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V

Abstract

In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.

Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha increased apoptosis of human endometrial cancer HEC-1A cells

“…Several studies have suggested that the lower left quadrant represents the surviving cells, the lower right quadrant represents the early apoptotic cells, the upper right quadrant represents the necrotic or postapoptotic cells, and the upper left quadrant represents the allowed detection error by using the annexin V-FITC/PI staining assay 14,15. In this study, we found that the downregulation of PGC-1α expression induced early apoptosis of HEC-1A cells.…”

Type I interferons are essential mediators of apoptotic death in virally infected cells

“…1a,b), a biochemical marker of apoptosis. In addition, we observed binding of annexin V-fluorescein isothiocyanate (FITC) to the outer leaflet of the plasma membrane, another early indicator of apoptosis (Vermes et al 1995) (Fig. 1c).…”

Platelet Mediated Complement Activation

“…Since PS is present on early apoptotic cells (Vermes, Haanen, Steffen-Nakken, and Reutelingsperger 1995), we propose that complement activation on microparticles may be involved in physiologic clearance mechanisms. Indeed, complement components C1q, Factor B, and C3 have all been shown to participate in the phagocytosis of apoptotic cells (Mevorach, Mascarenhas, Gershoev, and Eldon 1998).…”