Annexin V assay

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... 

Phosphatidylserine is a membrane phospholipid, which is normally restricted to the inner leaflet of the plasma membrane. The translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an early event in apoptosis. Annexin V, an endogenous human protein with a high affinity for membrane-bound phosphatidylserine, can be used in vitro to detect apoptosis. An annexin V assay in a microfluidic system has been developed for flow cytometric analysis of apoptosis using a minimal number of cells. 

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. 

Lecoeur, H., Prevost, M.C. and Gougeon, M.L., 2001, Oncosis is associated with exposure of phosphatidylserine residues on the outside layer of the plasma membrane A reconsideration ofthe specificity of the annexin V/propidium iodide assay. Cytometry, 44 65-72. 

This procedure, which complements other methods for distinguishing apoptotic and necrotic cell death, employs annexin V-PE as a marker for early apoptotic cells, and 7-AAD for late apoptotic or necrotic cells. Although other versions of this assay have used annexin V-fluorescein together with PI, that combination precludes the use of a third fluorescence color to measure an additional parameter, such as a phenotypic marker, because PI, unlike 7-AAD, has a broad emission spectrum that includes both orange and red fluorescence. 

In this study, annexin V/PI assay was used to detect cell death by flow cytometry (30) (see Note 11). 

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,... 

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). 

LIGHT SCATTER RHODAMINE 123, DiOC(6)3, or JC-1 with ANNEXIN V and PI with TUNEL ASSAY eg SNARF... 

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). 

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. 

Differential loss in cell membrane integrity between apoptotic and oncotic cells Combination assays Propidium iodide—YO-PRO-1 Acridine Orange-Ethidium Bromide Annexin V-Propidium iodide Useful for live, unfixed cells or unfixed organs only... 

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