Neutral Comet Assay to Detect and Quantitate DNA Double-Strand Breaksin Hematopoietic Stem Cells

In vertebrates, hematopoietic stem cells (HSCs) regulate the supply of blood cells throughout the lifetime and help to maintain homeostasis. Due to their long lifespan, genetic integrity is paramount for these cells, and accordingly, a number of stem cell-specific mechanisms are employed. However, HSCs tend to show more DNA damage with increasing age due to an imbalance between proliferation rates and DNA damage responses. The comet assay is the most common and reliable method to study DNA strand breaks at the single-cell level. This procedure is based on the electrophoresis of agarose-embedded lysed cells. Following the electrophoretic mobilization of DNA, it is stained with fluorescent DNA-binding dye. Broken DNA strands migrate based on fragment size and form a tail-like structure called "the comet," whereas intact nuclear DNA remains a part of the head of the comet. Since the alkaline comet assay fails to differentiate between single and double-strand breaks (DSBs), we used a neutral comet assay to quantitate the DSBs in HSCs upon aging and other physiological stresses. The protocol presented here provides procedural details on this highly sensitive, rapid, and cost-effective assay, which can be used for rare populations of cells such as HSCs. Graphical abstract: The neutral comet assay is an extremely useful tool that allows the detection and quantitation of double-strand DNA breaks at the single-cell level. The graphical abstract represents a flowchart for the neutral comet assay procedure.