Annexin V conjugates allow the identification of cell surface changes that occur early during the apoptotic process using flow cytometry. Early in the apoptotic process, phosphatidylserine 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. The binding of Annexin V to phosphatidylserine is calcium dependent, reversible, and very tight with a Kd of approximately 5 x 10-10 M.
General Comments:
Different cell types vary in their phosphatidylserine (PS) content, along with the amount of PS exposure on the cell surface after cell death. The following protocol is a guideline for getting started, however it may be necessary to adjust the concentration of the Annexin V-FITC. Typically a 1 to 100 dilution of the Annexin V-conjugate is appropriate, however dilutions of 1:10 up to 1:1000 may be needed.
When setting up an experiment, it is necessary to calibrate the flow cytometer to avoid spectral overlap between the two PMT channels. For each experiment, a set of treated cells should be processed unstained and stained separately with Annexin V-FITC and propidium iodide to define the boundaries of each population.