Annexin V, apoptosis assay

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

The combination of annexin V binding assay and the TUNEL method can reveal the presence of three subpopulations of apoptotic cells in tissues (1) annexin V-positive/TUNEL-negative cells which are in the early phase of apoptosis,... 

Plasier B, Lloyd DR, Paul GC, Thomas CR, Al-Rubeai M (1999), Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay, J. Immunol. Methods 229 81-95. 

CLARKE R G, LUND E K, JOHNSON I T aud PINDER A c (2000) Apoptosis cau he detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, hut uot hy TUNEL assay or suh-GO DNA content . Cytometry, 39 141-50. 

V8. Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C. R, A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods 184, 39-52 (1995). 

Annexin V Annexin V is a molecule that binds to phosphatidylserine and, therefore, if conjugated to a fluorochrome, will identify apoptotic cells (which express phosphatidylserine on their surface). In the assay for apoptosis, annexin V must be used in conjunction with propidium iodide in order to exclude dead cells (which express phosphatidylserine on the internal side of their cytoplasmic membranes). 

A number of methods for the study of apoptosis and necrosis by flow cytometry have been developed (see review by Steensma et al., 2003). Unfortunately, none of the methods are rigorously specific for apoptosis and many show poor overall specificity for cell death or cannot discriminate between the terminal stages of apoptosis and necrosis. Moreover, some of the fluorochromes used to detect apoptosis have emission spectra that fully overlap the spectra of those typically used for immunophenotyping or have a broad emission spectrum that makes compensation in multi- or polychromatic flow cytometry difficult, if not impossible. Thus, the preferred method would be one that measures two aspects of apoptosis, can discriminate between apoptotic and dead cells, and uses fluorochromes that allow simultaneous immunophenotyping. One such method is the annexin-V/7-amino-actinomycin-D assay described by Merchant et al. (2001) that is available in kit from BD Biosystems (San... 

Caliper Technologies and Agilent have developed an annexin V assay in a microfluidic system, which allows flow cytometric analysis of apoptosis with a minimal number of cells [6, 7]. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by two-channel fluorescence detection. As only a small number of cells (as few as 50-100) are consumed per assay, this setup is particularly suitable for working with cells of limited availability, for example, primary cells. The system has been applied to evaluate staurosporine-induced apoptosis and annexin V binding in human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs). The results are in good... 

Buhlmann C, Valer M, Mueller O (2004) Combination of DNA laddering and annexin-V and caspase assays on one system - multiple apoptosis parameters analysis with a microfluidic chip-based system. Tissue Antigens 64 392-392... 

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