Background

Maintaining DNA integrity is a pivotal prerequisite for cells to ensure that all physiological processes function immaculately. Cells are constantly exposed to a plethora of exogenous and endogenous agents that can damage their DNA. Examples of exogenous noxious agents include ultraviolet light, ionizing radiation, and reactive chemical compounds, while endogenous damage can arise for example due to reactive oxygen- and nitrogen species derived from cellular metabolism (van Loon et al., 2010). If left unrepaired, damage to DNA can lead to mutations, which can, in turn, alter the functionality of the affected DNA. This potentially results in altered protein abundance or activity, thereby giving rise to processes such as cancer, neurodegeneration, and ageing (Hoeijmakers, 2009; Markkanen, 2017). To counteract the deleterious effects of DNA damage, cells have evolved a series of intricate DNA repair mechanisms that detect and repair such insults (Ciccia and Elledge, 2010; Jackson and Bartek, 2009). Given the relevance of DNA damage for disease, it is of considerable interest to be able to measure levels of DNA damage that are induced by exposure of cells to particular agents, or to assess whether the repair kinetics of such damage is altered through specific treatments or genetic backgrounds. Methods to measure DNA damage and repair range from PCR-based methods and enzyme-linked immunosorbent assays, all the way to more elaborate next-generation sequencing based methods (Li and Sancar, 2020). Among this multitude of assays, the comet assay is a widely used method to measure both the amount of DNA damage as well as its repair in cells (Collins, 2014; Olive, 2009). For this, cells are treated with DNA damaging agents and embedded into a thin layer of agarose on top of a microscope slide. Subsequent lysis removes all protein and lipid components to leave the so-called ‘nucleoids,’ which consist of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates towards the anode. The migrating DNA, depending on its degree of fragmentation, creates shapes its degree of fragmentation and creates shapes resembling comets, which can subsequently be visualized and analysed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, and both single-strand DNA breaks as well as double-strand breaks. In this protocol, we describe in detail how to perform the neutral comet assay to assess double-strand breaks and their repair in cultured human cell lines. This exact protocol was used in our recent publication (Clementi et al., 2020). For a detailed description of the alkaline comet assay that can be used to detect single-strand DNA breaks, please refer to the separate protocol that is published in Bio-protocol (Clementi et al., 2021).