Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Abstract

Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or twophoton microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.

Effect of Lipopolysaccharide Mediating Early- and Late- Activated THP-1 Macrophages on ECV304 Endothelial Cell Dysfunction: Dysregulation of Secretion of VEGF and Proliferation and Migration of ECV304

“…Two types of effector/target contacts were seen: either effector cell protrusions were pushed deep into pouches and lacunae of the target cell surface, or a wide area of intimate cell-to-cell contact was formed [31]. The appearance of anionic phosphatidylserine on the cell surface is the most remarkable feature in apoptosis because of the abundance of this phospholipid in membranes, its negative charge and the ability to change interactions with other lipids leading to disruption of lipid rafts [32]. This may be one of the mechanism of cellular contact-induce apoptosis in present study and the corresponding research will be carried out in the further.…”

Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep

“…For the detection of apoptosis and necrosis, the assessment was applied on the same aliquots that were used for the immunolabeling of FSHR with a double-direct method, as previously described (Riccardi & Nicoletti 2006, Demchenko 2013. Briefly, after washing the cells with the prescribed annexin V phospholipid-binding, calcium-dependent buffer, a MAB to annexin V conjugated to the fluorochrome phycoerythrin (PE) and the nucleic acid dye 7-amino-actinomycin (7-AAD) (BD Biosciences, Perth, WA, Australia) were added for a final concentration of 5 ml/1!10 6 cells.…”

“…All of the graphs for apoptosis and necrosis were based on Q2, which indicates a combination of apoptosis and necrosis. Q1 was not reliable because the positive stain for early apoptosis may have been induced during cell preparation and therefore would not be a true indication of cell health status (Amsterdam et al 2003, Demchenko 2013). …”

Sensing Inside the Living Cells

“…The methods based on molecular recognition of surface-exposed PS and PE allow to identify and characterize apoptotic cells (Demchenko 2013 ). The most popular of them are based on the property of the protein annexin V to interact with exposed PS in a Ca 2+ -dependent manner.…”