Fast Detection of DNA Damage

Hong Zhao, Zbigniew Darzynkiewicz

Extensive DNA fragmentation that generates a multitude of DNA double-stand breaks (DSBs) is a hallmark of apoptosis. We developed several variants of the widely used TUNEL methodology that is based on the use of exogenous terminal deoxynucleotidyl transferase (TdT) to label 3'OH ends in DSBs with fluorochromes. Flow- or image-cytometry is then employed to detect and quantify apoptotic cells labeled this way. Here, we describe a variant of this technique using BrdUTP as a TdT substrate. The incorporated BrdU is subsequently visualized by a fluorochrome-tagged antibody. This is a particularly simple, rapid, and sensitive approach to detect DSBs.We also describe modifications of the labeling protocol permitting the use of deoxyribonucleotides other than BrdUTP to label DSBs. Concurrent differential staining of cellular DNA and multiparameter analysis of cells by flow- or image-cytometry enable correlations between apoptosis induction and the cell cycle phase. Examples of the detection of apoptotic cells in cultures of human leukemic cell lines treated with TNF-α and DNA topoisomerase I inhibitor topotecan are presented. The protocol can be applied to cells treated with cytotoxic drugs in vitro, ex vivo, or to clinical samples.