Measuring DNA Damage Using the Alkaline Comet Assay in Cultured Cells

Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA-damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity. Given the relevance of DNA damage and its repair in disease, measuring the amount of both aspects is of considerable interest. The comet assay is a widely used method that allows the measurement of both DNA damage and its repair in cells. 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 so-called 'nucleoids' consisting of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates toward the anode depending on its degree of fragmentation and creates shapes resembling comets, which can be subsequently visualized and analyzed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, in addition to both DNA single-strand and double-strand breaks. In this protocol, we describe in detail how to perform the alkaline comet assay to assess single-strand breaks and their repair using cultured human cell lines. We describe the workflow for assessing the amount of DNA damage generated by agents such as hydrogen peroxide (H₂O₂) and methyl-methanesulfonate (MMS) or present endogenously in cells, and how to assess the repair kinetics after such an insult. The procedure described herein is easy to follow and allows the cost-effective assessment of single-strand breaks and their repair kinetics in cultured cells.