Oxidative Stress Group, Dept. Cell Biology, Microbiology and Molecular Biology, College of Arts and Sciences, University of South Florida.
Cepheid (Danaher Corp.), US Technical Operations.
J Vis Exp. 2022 May 10(183). doi: 10.3791/63559.
Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.
细胞不断受到来自内部和外部环境的各种因素的影响，这些因素可能会损害 DNA。这种损伤会导致细胞功能异常，因此 DNA 损伤可能在所有重大人类疾病（例如癌症、神经退行性疾病和心血管疾病以及衰老）的发生中起着关键作用。单细胞凝胶电泳（即彗星试验）是研究广泛类型 DNA 损伤（例如单链和双链断裂、碱易损位点、DNA-DNA 交联以及与某些修复酶结合时氧化嘌呤和嘧啶）形成和修复的最常用和最敏感的方法之一，无论是在体外还是体内系统中。然而，传统检测方法的低样品通量和繁琐的样品处理是限制其尽可能广泛应用的因素。随着彗星“评分”的日益自动化，现在的限制因素是处理大量彗星载玻片的能力。在这里，开发了一种高通量（HTP）彗星试验变体（HTP 彗星试验），该变体可显著增加分析的样品数量，缩短试验运行时间，减少单个载玻片操作次数、试剂需求以及凝胶物理损伤的风险。此外，由于载玻片的垂直方向和整体冷却，电泳槽的足迹显著减小。本文还报道了一种新颖的彗星试验载玻片冷却方法，该方法方便高效地促进了彗星凝胶的凝固。这里描述了这些设备在代表性彗星试验方法中的应用。这些简单的创新大大支持了彗星试验的应用，并将其应用于暴露生物学、生态毒理学、生物监测、毒性筛选/测试以及发病机制研究等领域。
