Background

During every round of cell proliferation, the genomic content is faithfully replicated, albeit with some errors. These errors lead to changes or modifications in the structure and chemical nature of genomic DNA. The replication machinery is tightly associated with DNA damage repair (DDR) pathways, thereby recovering cells from such damage (Chatterjee and Walker, 2017). Alternatively, the DNA damage response pathways can induce apoptosis (Nowsheen and Yang, 2012); however, some DNA damage is undetected and unrepaired, accumulating over a period of time. Accumulation of this DNA damage during aging poses a serious challenge to the healthy functioning of a variety of tissues, including hematopoietic systems. Recent studies have indicated that accumulation of age-related physiological changes in the stem cell population leads to alterations in immune system function (Oh et al., 2014). In addition, several age-associated malignancies have been linked to DNA damage accumulation in the stem cell compartment (Beerman, 2017). Hematopoietic stem cells have cytoprotective and genoprotective mechanisms to safeguard their long-lasting functional potential; however, it has been demonstrated that DNA damage accumulates in HSCs during the aging process. Several research groups have proposed that this accumulation is due to increased proliferation rates in old HSCs. In contrast, others show that long-term dormancy may prevent rare DNA damage from accumulating due to lack of replication-associated repair (Beerman et al., 2014).

To understand the fundamental processes involved in DNA damage accumulation and repair, quantitation of DNA damage is vital (Lu et al., 2017). In 1984, Ostling and Johanson demonstrated the electrophoretic mobility of DNA fragments from the core nuclei (Ostling and Johanson, 1984). When single cells embedded in low-melting-point agarose are electrophoresed, the DNA-damaged fragments move faster, giving rise to tail-like structures resembling a comet. The intact part of the DNA is called the head of the comet, and the trail produced due to impaired DNA is known as the tail of the comet. This single-cell gel electrophoresis assay, also known as the comet assay, is one of the most reliable and commonly used methods for analyzing DNA damage in cells. It has been successfully applied to understand the processes involved in genotoxicity, molecular epidemiology, and human bio-monitoring, along with solving basic biology questions regarding DNA damage and repair (Collins, 2004). This technique was further modified by Singh et al., who showed a substantial increase in reproducibility under alkaline conditions (Lu et al., 2017). At alkaline pH, the sensitive sites are knocked down and the strands break due to distortion in their conformation. Therefore, the alkaline comet assay is commonly used to analyze strand breaks, DNA-protein crosslinks, and alkali-labile sites; nevertheless, the specificity for double-strand breaks is compromised (Lu et al., 2017). To overcome this issue, the neutral comet assay was further developed (Calini et al., 2002); it protects the DNA double strands from distortion, thus aiding in the specific detection of double-strand DNA damage. These DNA strand breaks result in the lengthening of DNA loops in genomic DNA, which form a comet-like tail in an electrophoretic mobility assay (Cortes-Gutierrez et al., 2012). Because double-strand DNA breaks lead to mutations and chromosomal abnormalities, it is important to detect and quantitate them. The neutral comet assay is of immense importance and is highly efficient for in vivo and ex vivo systems (Sharma et al., 2011). The comets formed due to electrophoretic mobility can be visualized by fluorescence microscopy. The percentage of DNA damage can be estimated from the percentage of DNA present in the comet tail since they are directly proportional to one other. With advancements in technology such as fluorescent staining and automated comet scoring software, the comet assay has become popular for detecting DNA damage (Beedanagari, 2017). In the case of rare cell populations such as HSCs, the neutral comet assay has emerged as a promising technique to study DNA damage responses due to physiological, pathological, and age-associated stresses (Hazlehurst and Dalton, 2001; Milyavsky et al., 2010). The present protocol describes the method to assess DNA damage in freshly sorted hematopoietic stem cells from mice bone marrow.