Abstract
Single-cell gel electrophoresis (SCGE or Comet assay) and the Fast Halo assay, also known as the Halo assay, are powerful tools to generate DNA damage measurements with single-cell resolution. Though these techniques are prone to have variability, they can be robust tools for quantifying DNA damage when planned and executed carefully. Here, we present both assays and highlight each technique’s advantages and challenges in measuring DNA damage in cells with limiting cell number, such as hematopoietic stem cells (HSCs). The Comet assay is highly sensitive at the cost of increased variability. The Halo assay attenuates some of the effects of variability present in the Comet assay but does not eliminate them entirely and is less sensitive. Overall, the Comet and Halo assays are powerful means of directly measuring DNA damage. We recommend the below methods for detecting damage in hematopoietic stem cells, but the methods can easily be adjusted for measuring damage in any type of single cells in suspension.
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References
Wang J, Sun Q, Morita Y, Jiang H, Gross A, Lechel A, Hildner K, Guachalla LM, Gompf A, Hartmann D, Schambach A, Wuestefeld T, Dauch D, Schrezenmeier H, Hofmann WK, Nakauchi H, Ju Z, Kestler HA, Zender L, Rudolph KL (2012) A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage. Cell 148(5):1001–1014. https://doi.org/10.1016/j.cell.2012.01.040
Yahata T, Takanashi T, Muguruma Y, Ibrahim AA, Matsuzawa H, Uno T, Sheng Y, Onizuka M, Ito M, Kato S, Ando K (2011) Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells. Blood 118(11):2941–2950. https://doi.org/10.1182/blood-2011-01-330050
Rossi DJ, Bryder D, Zahn JM, Ahlenius H, Sonu R, Wagers AJ, Weissman IL (2005) Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A 102(26):9194–9199. https://doi.org/10.1073/pnas.0503280102
Beerman I, Seita J, Inlay MA, Weissman IL, Rossi DJ (2014) Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle. Cell Stem Cell 15(1):37–50. https://doi.org/10.1016/j.stem.2014.04.016
McNeely T, Leone M, Yanai H, Beerman I (2019) DNA damage in aging, the stem cell perspective. Hum Genet. https://doi.org/10.1007/s00439-019-02047-z
Vijg J (2000) Somatic mutations and aging: a re-evaluation. Mutat Res 447(1):117–135. https://doi.org/10.1016/s0027-5107(99)00202-x
Kohn KW, Erickson LC, Ewig RA, Friedman CA (1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15(21):4629–4637. https://doi.org/10.1021/bi00666a013
Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123(1):291–298. https://doi.org/10.1016/0006-291x(84)90411-x
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191. https://doi.org/10.1016/0014-4827(88)90265-0
Sestili P, Calcabrini C, Diaz AR, Fimognari C, Stocchi V (2017) The Fast-Halo assay for the detection of DNA damage. Methods Mol Biol 1644:75–93. https://doi.org/10.1007/978-1-4939-7187-9_6
Sestili P, Martinelli C, Stocchi V (2006) The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single-cell level. Mutat Res 607(2):205–214. https://doi.org/10.1016/j.mrgentox.2006.04.018
Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, Kruszewski M, Smith CC, Stetina R (2008) The comet assay: topical issues. Mutagenesis 23(3):143–151. https://doi.org/10.1093/mutage/gem051
Collins AR, Ma AG, Duthie SJ (1995) The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat Res 336(1):69–77. https://doi.org/10.1016/0921-8777(94)00043-6
Forchhammer L, Johansson C, Loft S, Moller L, Godschalk RW, Langie SA, Jones GD, Kwok RW, Collins AR, Azqueta A, Phillips DH, Sozeri O, Stepnik M, Palus J, Vogel U, Wallin H, Routledge MN, Handforth C, Allione A, Matullo G, Teixeira JP, Costa S, Riso P, Porrini M, Moller P (2010) Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial. Mutagenesis 25(2):113–123. https://doi.org/10.1093/mutage/gep048
Collins AR, El Yamani N, Lorenzo Y, Shaposhnikov S, Brunborg G, Azqueta A (2014) Controlling variation in the comet assay. Front Genet 5:359. https://doi.org/10.3389/fgene.2014.00359
Maurya DK (2014) HaloJ: an ImageJ program for semiautomatic quantification of DNA damage at single-cell level. Int J Toxicol 33(5):362–366. https://doi.org/10.1177/1091581814549961
Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121(7):1109–1121. https://doi.org/10.1016/j.cell.2005.05.026
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This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging.
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Ayyar, S., Beerman, I. (2023). Detection of DNA Damage in Hematopoietic Stem Cells. In: Pelus, L.M., Hoggatt, J. (eds) Hematopoietic Stem Cells. Methods in Molecular Biology, vol 2567. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2679-5_2
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