Detection of end-stage apoptosis by ApopTag® TUNEL technique

DNA fragmentation, the end stage of apoptosis, is the measure of ultimate demise of the cell. A convenient method for examining apoptosis via DNA fragmentation is by the Terminal deoxynucleotidyl transferase (Tdt) dUTP Nick-End Labeling (TUNEL) assay where the DNA strand breaks are detected by enzymatically labeling the free 3'-OH termini with modified nucleotides. ApopTag(®) kits detect single-stranded and double-stranded breaks associated with apoptosis. This technique is also helpful to distinguish between apoptotic and necrotic cell death where the latter is associated with random DNA fragment lengths producing a DNA smear. Apoptotic cells stained positive with ApopTag(®) kits are easier to detect and their identification is more certain, as compared to the examination of simply histochemically stained tissues. In addition, quantitative results can be obtained using flow cytometry and apoptotic cells can be differentiated from necrotic cells with greater than tenfold sensitivity.