Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Abstract

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.

Role of the Phosphatidylserine Receptor TIM-1 in Enveloped-Virus Entry

“…In order to determine if TIM-1 was binding to PtdSer on virions, we initially determined if PtdSer was present in pseudovirion membranes by ELISA studies using purified recombinant HA-tagged AnxV. AnxV is a cellular protein that binds to PtdSer (52) and detects PtdSer on the surface of apoptotic cells (53). AnxV bound to EBOV pseudovirion-coated ELISA plates in a dose-dependent manner but not to plates lacking pseudovirions (Fig.…”

Cytometric Analysis of Cytotoxicity of Polyphenols and Related Phenolics to Rat Thymocytes: Potent Cytotoxicity of Resveratrol to Normal Cells

“…This suggestion is partly supported by the finding that resveratrol led to an increase in the population of living cells with exposed phosphatidylserine ( fig. 5), which is an indicator of the early stage of apoptosis [19]. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone did not completely inhibit the resveratrol-induced increase in the population of cells with hypodiploidal DNA, and N-acetyl-leucyl-leucyl-norleucinal did not have any effect on this increase.…”

Detection of apoptosis induced by new type gosling viral enteritis virus in vitro through fluorescein annexin V-FITC/PI double labeling

“…This research indicated that NGVE virus recognized as an adenovirus [17,19,20] could induce DEF undergoing apoptosis, which has never been reported before. FACS is frequently used to monitor early apoptosis [26][27][28][29] , which should always be confirmed by the inspection of cells under electron or fluorescence microscope. Annexin V-FITC positive cells were first observed in NGVEVinfected DEF cells at 72 h p.i.…”

“…In the early stage of apoptosis, which occurs at the cell surface, 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 [25][26][27] . Annexin V-FITC is a phospholipid-binding protein with a high affinity for PS, which can be used as a sensitive probe for PS exposure to the cell membrane [28][29][30] . However, it has also been reported that it binds to the inner face of the plasma membrane that has lost its integrity during the late stage of apoptosis, also known as secondary necrosis [31] .…”