CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells
CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells
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DOI：
10.3791/50607
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发表时间：
2014-10-01
影响因子：
1.2
通讯作者：
Engelward, Bevin P.
中科院分区：
文献类型：
--
作者：
Ge, Jing;Prasongtanakij, Somsak;Engelward, Bevin P.
DNA损伤剂可以促进衰老、疾病和癌症，它们在环境中普遍存在，并作为正常细胞代谢产物在人体细胞内产生。具有讽刺意味的是，高剂量的DNA损伤剂也用于治疗癌症。因此，量化DNA损伤反应的能力在公共卫生、制药和临床领域中至关重要。在这里，我们描述了一种新的平台，利用微加工技术，在一个固定的微阵列图案细胞。"CometChip“是基于成熟的单细胞凝胶电泳测定法（a.k.a.彗星试验），其通过评估电场中DNA通过基质迁移的程度来估计DNA损伤的水平。通过该测定测量的损伤类型包括脱碱基位点、交联和链断裂。在传统的测定中，细胞不是随机分散在琼脂糖中，而是通过重力捕获到琼脂糖微孔阵列中。该平台还可以从标准显微镜载玻片的尺寸扩展到96孔格式，从而实现并行处理。在这里，我们描述了使用芯片来评估已知的遗传毒性剂引起的DNA损伤和暴露后的细胞修复反应的协议。通过生物学和工程学原理的整合，该方法增强了对人类细胞中DNA损伤的稳健和灵敏的测量，并为遗传毒性测试、药物开发、流行病学研究和临床测定提供了必要的通量。
DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The 'CometChip' is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.