NYMC Faculty Publications
Rapid Detection of DNA Strand Breaks in Apoptotic Cells by Flow- and Image-Cytometry
Journal Title
Methods in Molecular Biology
First Page
139
Last Page
149
Document Type
Article
Publication Date
7-15-2017
Department
Pathology, Microbiology and Immunology
Abstract
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-alpha 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.
Recommended Citation
Zhao, H., & Darzynkiewicz, Z. (2017). Rapid Detection of DNA Strand Breaks in Apoptotic Cells by Flow- and Image-Cytometry. Methods in Molecular Biology, 1644, 139-149. https://doi.org/10.1007/978-1-4939-7187-9_12
