Apoptosis Measurement by Annexin V Staining

Several methods exist for the detection of apoptosis using features of the cell as it undergoes the various stages leading to the death of the cell (1 ,2 ). One of the earliest uses of flow cytometry was the detecting of a sub-G₀ peak in the DNA histogram that showed fragmentation of the nucleus (3 ). The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay also detects fragmentation of DNA by labeling the broken ends of DNA sequences with fluorescein isothiocynate conjugate (FITC)-labeled nucleotides (4 ,5 ). Unfortunately, DNA fragmentation occurs at a late stage in the process of apoptosis (6 ) and is not specific to apoptotic cells. Necrotic cells can also produce these appearances in a flow-cytometric assay. Recently, PARP cleavage (7 ) and annexin V (8 –11 ) have claimed more specificity. PARP cleavage occurs early in the response as a result of the activity of caspase-3 and correlates well with chromatin condensation (12 ). PARP cleavage is associated with the condensed chromatin in apoptotic cells as a measure of apoptosis. It may appear as early as 3 h post the apoptosis inducing event and precedes the ability to detect actual DNA fragmentation. The annexin V assay has been widely accepted as a marker of apoptosis (13 –15 ) and the remainder of this chapter will be devoted to its measurement by flow cytometry.