UNIT 3.12 Alkaline Comet Assay for Assessing DNA Damage in Individual Cells Xinzhu Pu, 1 Zemin Wang, 2 and James E. Klaunig 2 1 Biomolecular Research Center, Boise State University, Boise, Idaho 2 Department of Environmental Health, Indiana University, Bloomington, Indiana Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali- labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. C  2015 by John Wiley & Sons, Inc. Keywords: comet assay  DNA strand break  oxidative DNA damage  genotoxicity How to cite this article: Pu, X., Wang, Z., and Klaunig, J.E. 2015. Alkaline comet assay for assessing DNA damage in individual cells. Curr. Protoc. Toxicol. 65:3.12.1-3.12.11. doi: 10.1002/0471140856.tx0312s65 INTRODUCTION Many environmental agents can damage the hereditary materials of living organisms and lead to a wide range of disorders in experimental animals and humans (Brusick, 2001). A variety of methods have been developed for assessing genotoxicity. The single-cell gel electrophoresis assay, commonly known as a comet assay, is a simple and sensitive technique for the detection of DNA damage and repair in eukaryotic cells (Singh et al., 1988; Tice et al., 2000). This unit describes the procedures for measuring DNA strand breaks and oxidative damage by a comet assay performed under alkaline conditions. Several variations of the comet assays have been developed to detect different types of DNA damage. The most widely used version is the alkaline comet assay, which is performed under high pH (>13). It is recognized that the alkaline comet assay is the optimal version for assessing agents with genotoxic activity (Tice et al., 2000). The alkaline comet assay is able to detect single-strand breaks (SSBs), double-strand breaks (DSBs), and alkaline-labile sites (ALS) in DNA. Incorporation of lesion-specific, oxidative DNA damage repair enzymes (e.g., formamidopyrimidine-DNA glycosylase; Fpg) in the alkaline comet assay procedure provides a simple and sensitive method for the measurement of oxidative DNA damage (Smith et al., 2006; Collins, 2009). The Basic Protocol describes the alkaline comet assay for the detection of DNA strand breaks and oxidative DNA damage. Current Protocols in Toxicology 3.12.1-3.12.11, August 2015 Published online August 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471140856.tx0312s65 Copyright C  2015 John Wiley & Sons, Inc. Genetic Toxicology 3.12.1 Supplement 65