Analysis of apoptosis by cytometry using TUNEL assay

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3′-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4′,6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase.