Annexin-V-FITC

A two-color dot plot (Fig. 8.23) of cells stained with propidium iodide and annexin V FITC will indicate cells in three of the four quadrants. Unstained cells are alive and well and are the double negatives they neither express phosphatidylserine on their surface nor take up propidium iodide through leaky membranes. Cells that stain just with annexin V are apoptotic they have begun to express phosphatidylserine on their surface, but have not yet gone through the process that leads to permeabilization of their cytoplasmic membrane. Cells that stain both with propidium iodide and annexin V are necrotic (that is, dead) they take up propidium iodide and also stain with annexin Y. With a permeable cell, the flow cytometer cannot tell us whether the annexin V is on the outside of the membrane (because the cells have gone through apoptosis before membrane permeabilization) or on the inside of the membrane (because the cells have died by the necrotic pathway without apoptosis). 

Therefore, by staining cells with a combination of annexin V-FITC and PI, it is possible to distinguish cells at different stages of apoptosis. 

Annexin V-FITC solution Dissolve annexin V-FITC conjugate (1 1 stoichiometric complex, available from BRAND Applications, The Netherlands) in... 

With a Pasteur pipet, gently remove the culture medium in which the cells were growing (or PBS is they were attached electrostatically) and immediately (without allowing cells to dry) replace it with the Annexin V-FITC solution. Cover with a 2 x 4 cm strip of Parafilm and place in the box containing wet tissue or filter paper to ensure 100% humidity. 

With a Pasteur pipet, gently remove the Annexin V-FITC solution and immediately mount the cells under a coverslip in a drop of the staining solution of PI. 

Figure 11.8 (a) Cell viability and (b) LDH leakage of Vero cells treated with p-G and f-G at different concentrations, (c) Flow cytogram showing apoptosis assay based on annexin V-FITC and PI staining of cells. Reproduced from ref. 142 with permission from the Royal Society of Chemistry. 

Fluorescein isothiocyanate (FITC)-conjugated annexin V is available from many different suppliers. In the present study, it was produced in house as previously described (30). Protect from light and store at +4°C. 

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. 

Membrane asymmetry changes can be detected by flow cytometry using a fluorescent marker (e.g. fluorescein, FITC) conjugated to annexin V, a protein that has high affinity for phosphatidylserine. When using a fluorescence microscope, this technique can be quantitative if a hemocyt-ometer is used. 

Annexin V preferentially binds to PS, and can be used as an early indicator of apoptosis using flow cytometry or in situ detection. Annexin V conjugated to either FITC or to biotin can be used for the detection of cell surface changes during apoptosis. In addition, propidium iodide can be used on unfixed samples to determine the population of cells that have lost membrane integrity, an indication of late apoptosis or necrosis. 

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