安徽医科大学学报2024，Vol.59Issue(2)：219-223,5.DOI:10.19405/j.cnki.issn1000-1492.2024.02.006
彗星实验方法的优化及影响因素的分析
Optimization of comet experiment method and analysis of influencing factors
摘要
Abstract
Objective To detect cell DNA damage more effectively by optimizing the influencing factors of Comet assay and establishing a positive control.Methods Based on the traditional alkaline comet assay,the gel prepara-tion method,gel concentration,agarose solvent,lysis time,cell density and electrophoresis voltage were opti-mized.HL-60 cells were induced by hydrogen peroxide(H2O2)as a DNA damage inducer as a positive control.Results The optimized experimental results showed that the gel preparation method improved from three-step meth-od to two-step method.PBS was used as agarose solvent,0.8％normal melting point agarose was used to prepare the bottom layer,and 0.7％low melting point agarose was mixed with cell suspension as the second layer.The gel preparation effect was better,the operation was simpler,the time was saved,and the degumming problem was bet-ter solved.After lysis for 1 h and electrophoresis at 1 V/cm voltage,representative comet images were obtained.The results were easy to interpret and could effectively avoid false positive results.The positive control of comet as-say was successfully established by comparing the DNA damage of HL-60 cells induced by different concentrations of H2 O2.Conclusion Compared with the traditional method,the optimized comet assay method is simpler,faster,more accurate,lower cost and has better repeatability,which can quickly detect DNA damage in cells.关键词
彗星实验/HL-60/DNA损伤模型Key words
comet assay/HL-60/DNA damage model分类
生物科学引用本文复制引用
范南英,张鹏..彗星实验方法的优化及影响因素的分析[J].安徽医科大学学报,2024,59(2):219-223,5.基金项目
国家自然科学基金(编号:81960036) （编号:81960036）
贵州省科技计划项目(编号:黔科合基础-ZK[2021]重点005) （编号:黔科合基础-ZK[2021]重点005）
贵州省教育厅高校科技创新团队(编号:黔教技[2023]068) （编号:黔教技[2023]068）
