Annexin V Assays
The coupling of annexin to fluorescein (Annexin V-FITC), or biotin (Annexin V-Biotin), generates a direct, rapid, and simple method for the detection of apoptosis on unfixed cells by microscopy or flow cytometry. Some of the earliest apoptotic changes occur at the cell surface. One of the better understood cell surface modifications is exposure of phosphatidylserine (PS). Normally, PS is confined to the inner layer of the lipid bilayer. This asymmetric distribution is maintained in normal cells by the action of specific proteins. After induction of apoptosis under the influence of translocases, the PS is flipped from the inner to the outer bilayer, rendering the molecule available for detection. Annexin V is a blood clotting factor that exhibits a high calcium-dependent specificity for PS binding.
Annexin V Kits (2)
Early in the apoptosis process, phosphatidylserine (PS) becomes exposed on the cell surface by flipping from the inner to outer leaflet of the cytoplasmic membrane. This event is thought to be important for macrophage recognition of cells undergoing apoptosis, thus allowing the cells to be rapidly phagocytosed. The binding of Annexin V to phosphatidylserine is calcium-dependent, reversible, but very tight, with a Kd of approximately 5 x 10-10 M. At low PS concentration, a binding ratio of 8 annexins to one PS has been reported. These features make Annexin V conjugates ideal for identifying membrane changes associated with apoptosis, by using either flow cytometric or in situ labeling methods. TACS® Annexin V kits are supplied with an Annexin V conjugate, an optimized binding buffer and propidium iodide. Cells are harvested and washed, then incubated with an Annexin V conjugate in binding buffer, for 15 minutes at room temperature prior to direct analysis. Alternatively, a secondary streptavidin conjugate can be used for detection. Propidium iodide is included in the incubation mix to identify cells that have lost membrane integrity (i.e. late apoptotic/necrotic cells). As cells disintegrate, greater access to the inner cell membrane allows for additional Annexin V binding. Thus, double labeling is used to help differentiate between early and late apoptotic/necrotic events.
Annexin V Reagents (4)Annexin V related reagents are also available for purchase individually.