Epigenetics and Stem Cell Unit, Translational Gerontology Branch, National Institute on Aging, Baltimore, MD, USA.
Methods Mol Biol. 2023;2567:11-28. doi: 10.1007/978-1-0716-2679-5_2.
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.
单细胞凝胶电泳 (SCGE 或彗星分析) 和 Fast Halo 分析，也称为 Halo 分析，是一种强大的工具，可以实现单细胞分辨率的 DNA 损伤测量。尽管这些技术容易产生变异性，但如果仔细规划和执行，它们可以成为量化 DNA 损伤的可靠工具。在这里，我们介绍了这两种检测方法，并强调了每种技术在测量具有有限细胞数量的细胞(如造血干细胞 [HSCs])中的 DNA 损伤方面的优势和挑战。彗星分析在增加变异性的代价下具有高度的敏感性。Halo 分析减轻了彗星分析中存在的一些变异性影响，但并没有完全消除它们，而且敏感性较低。总的来说，彗星分析和 Halo 分析是直接测量 DNA 损伤的有力手段。我们建议以下方法用于检测造血干细胞中的损伤，但这些方法可以很容易地调整用于测量悬浮的任何类型的单细胞中的损伤。
