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Technical Updates to the Comet Assay In Vivo for Assessing DNA Damage in Zebrafish Embryos from Fresh and Frozen Cell Suspensions
Zebrafish ( IF 2 ) Pub Date : 2020-06-12 , DOI: 10.1089/zeb.2020.1857
Carla Martins 1 , Pedro M. Costa 1
Affiliation  

The Single-Cell Gel Electrophoresis, simply known as the Comet assay, is a sensitive and quick technique used to quantitate DNA damage, widely used to assess the effects of genotoxicants and mutagens in animal cells. Still, performing the assay on peripheral or cultured cells is far more expeditious and cost effective than solid tissue, especially from small biological model like the zebrafish embryo. The current work describes and validates a highly cost-effective protocol of the updated Comet assay designed for zebrafish embryos. Compared with the few previous applications of the Comet assay on this biological model, the present method successfully simplifies the process of cell harvesting and resuspending, producing a much higher yield of viable nucleoids with reduced basal DNA damage, even from a small number of embryos, and compatible with scoring with safe fluorescent dyes. Additionally, the protocol can be just as easily performed on freshly harvested cells of cells frozen in dimethyl sulfoxide (DMSO)-containing physiological buffer, without a significant increase of DNA damage, which is another highly relevant update, especially for researchers handling high numbers of samples.

中文翻译:

用于评估新鲜和冷冻细胞悬浮液对斑马鱼胚胎DNA损伤的体内彗星检测技术更新

单细胞凝胶电泳法,简称为彗星分析法,是一种用于定量DNA损伤的灵敏,快速的技术,广泛用于评估遗传毒性剂和诱变剂在动物细胞中的作用。但是,与实体组织相比,对外围或培养的细胞进行测定要快捷得多,并且更具成本效益,尤其是从像斑马鱼胚胎这样的小型生物模型中。当前的工作描述并验证了为斑马鱼胚胎设计的更新彗星试验的高成本效益方案。与以前在此生物学模型上进行彗星分析的少数应用相比,本方法成功地简化了细胞收获和重悬过程,即使从少数胚胎中产生的基础核糖核酸损伤减少的情况下,产生的活核苷酸的产量也要高得多,并与使用安全的荧光染料评分兼容。此外,该协议可以在含有二甲基亚砜(DMSO)的生理缓冲液中冷冻的新鲜收获细胞上轻松进行,而不会显着增加DNA损伤,这是另一个高度相关的更新,特别是对于处理大量DNA的研究人员而言样品。
更新日期:2020-06-12
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