基于EdU/BrdU双标记法检测细胞周期动力学的方法  

Method for retermine cell-cycle dynamics based on EdU/BrdU dual labeling

作　　者：袁月 艾立丽 张鹏 Yuan Yue;Ai Lili;Zhang Peng(Dept of Biology,Basic Medical Sciences College,Guizhou Medical University,Guiyang 550004;Center for Tissue Engineering and Stem Cell Research,Key Laboratory of Functional Nucleic Acids-Based Biopharmaceutical Research,Guizhou Medical University,Guiyang 550004)

机构地区：[1]贵州医科大学基础医学院生物学教研室,贵阳550004 [2]贵州医科大学组织工程与干细胞实验中心,功能核酸生物药研究重点实验室,贵阳550004

出　　处：《安徽医科大学学报》2024年第12期2170-2175,2182,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学青年基金项目(编号:32000601);贵州省科技计划项目(编号:黔科合基础-ZK[2021]重点005);贵州省教育厅科技创新团队(编号:黔教计[2023]068号)。

摘　　要：目的建立一种基于5-乙炔基-2′-脱氧尿苷(EdU)和5-溴脱氧尿嘧啶核苷(BrdU)双标记法检测细胞周期S期时间(Ts)和总细胞周期时间(Tc)的方法。方法通过EdU和BrdU双重标记HEK293T细胞后进行免疫荧光染色,利用高内涵细胞成像分析仪采集荧光信号,统计整个细胞群中EdU阳性和BrdU阳性细胞个数,基于之前发表文章中的细胞周期计算公式,对其进行改进后,可精准计算出细胞的Ts和Tc时长,并利用建立的方法对人胚胎干细胞细胞周期时长进行计算。结果根据EdU/BrdU双标记的方法计算出人胚胎干细胞的Ts为10.9 h,Tc为15.3 h。结论基于EdU/BrdU双标记法能检测出细胞的Ts和Tc时长,是一种可以精准高效检测细胞周期的方法。Objective To establish a method for detecting the duration of the synthesis-phase(Ts)and the total cell-cycle(Tc)based on the dual labeling of 5-ethynyl-2′deoxyuridine(EdU)and 5-Bromodeoxyuridinc(BrdU).Methods HEK293T cells were double labeled with EdU and BrdU and then subjected to immunofluorescence staining.The fluorescence signals were collected using a High-Content Imaging System.The numbers of EdU-positive and BrdU-positive cells in the entire cell population were counted.Based on the cell cycle calculation formula in a previously published article,it was improved to accurately calculate the duration of Ts and Tc of cells.The cell cycle time of human embryonic stem cells was calculated using the established method.Results Based on the kinetic parameters of the EdU/BrdU dual labeling,the Ts of human embryonic stem cells was 10.9 h,and the Tc was 15.3 h.Conclusion The EdU/BrdU dual labeling method can be used to detect the duration of the cell cycle,providing an accurate and efficient method for cell cycle analysis.

关 键 词：EDU BRDU 细胞周期 免疫荧光 人胚胎干细胞 

分 类 号：Q2-33[生物学—细胞生物学]