Annexin V Apoptosis Assay

Translocalisation of phosphatidylserine (PS) from the inner to the outer membrane layer happens early in apoptosis and precedes other apoptotic events such as loss of membrane integrity and DNA fragmentation. Annexins are group of cellular proteins that bind to phospholipids in a calcium-dependent manner. A member of this group, Annexin V, has proven to be a useful tool in detecting apoptotic cells since it has a high affinity for PS.

Using fluorescently labeled Annexin V the externalization of PS can easily be detected. Annexin V may also bind to PS on late apoptotic and necrotic cells but as the membrane integrity on these cells has been lost, these cells can be distinguished from early apoptotic cells by the use of the impermeant dye propidium iodide.

Figure: Jurkat cells were grown in the absence (upper row) or in the presence (lower row) of camptothecin (CPT). Cells were stained with Hoechst-33342, Annexin V FITC conjugate and Propidium Iodide (PI) and analysed using the Annexin V Assay and a NucleoCounter^® NC-3000™. Scatter plots and histograms were obtained from the NucleoView™ NC-3000™ software. Quadrants and markers in the displayed plots were used to demarcate the various cell populations. In this example camptothecin causes a dramatic increase of early apoptoctic cells (Annexin V positive and PI negative cells).