流式细胞术检测细胞周期影响因素及不同免疫细胞亚群周期分析  

Factors Influencing Flow Cytometry-based Cell Cycle Detection and Analysis of Various Immune Cell Subpopulations

作　　者：刘丹[1] 张洁[1,2] 郭正阳[1] 薛丽香[1,2] 王羽晴 LIU Dan;ZHANG Jie;GUO Zheng-Yang;XUE Li-Xiang;WANG Yu-Qing(Center of Basic Medical Research,Institute of Medical Innovation and Research,Peking University Third Hospital,Beijing 100191,China;Biobank,Peking University Third Hospital,Beijing 100191,China)

机构地区：[1]北京大学第三医院医学创新研究院基础医学研究中心,北京100191 [2]北京大学第三医院生物样本库,北京100191

出　　处：《中国生物化学与分子生物学报》2024年第9期1308-1316,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.82203433,No.81901570);北京大学第三医院院临床重点项目(No.BYSY2022070)资助。

摘　　要：细胞周期检测对于了解细胞增殖状态、细胞功能研究、药物筛选与评估等领域具有重要意义。流式细胞仪分析细胞周期是目前较为经典且广泛应用的检测手段之一。碘化丙啶(propidium iodide,PI)单染是最常用的基于流式的检测细胞周期方法,然而,该方法在使用过程有诸多处理因素会对检测结果造成影响。此外,不同免疫细胞亚群在不同细胞周期阶段的分布差异也有助于研究免疫反应,了解疾病状态。本文以B16-F10细胞系为例,采用PI单染法,从固定条件、上机条件和软件分析3个角度,评价多种处理因素对细胞周期检测结果的影响。根据结果分析,在进行细胞周期检测时,建议取3×10^(6)个细胞,用300μL预冷PBS重悬细胞后逐滴滴入700μL预冷的无水乙醇,放置于4℃或-20℃过夜固定,低速收样,收样速率为每秒400~600颗粒数,去黏连后需收集至少3000个细胞。另外,通过EdU、PI双指标染色可以精确划分细胞周期,而细胞表面标志物染色联合Ki-67和PI染色法,可在不进行细胞分选的情况下进行不同免疫细胞亚群周期分析。本文较为系统的探讨了PI单染法检测细胞周期的影响因素,提供了标准的实验操作流程。建立了EdU联合PI检测细胞周期和针对不同免疫细胞亚群周期分析方案,拓宽了细胞周期检测方式。Cell cycle analysis is essential for determining the cell proliferation state,studying cell functions,and evaluating drug effects.Flow cytometry is a commonly used method for cell cycle detection,with propidium iodide(PI)being the most widely used fluorescein.Nevertheless,various factors may affect the test results.Additionally,comparing distributions of immune cell subpopulations across different cell cycle stages can provide valuable insights into immunological responses and disease conditions.In this research,the B16-F10 cell line was used to study the impact of three factors on PI staining-based cell cycle detection:fixation settings,sample preparation conditions,and software analysis.To fix cells,it is suggested to suspend 3×10^(6)cells in 300μL of pre-cooled PBS,add 700μL of 100%ethanol dropwise,fix overnight at 4℃or-20℃,and collect at a low flow rate(400-600 events/s)to ensure collection of at least 3000 singlets.Furthermore,dual-labeling with 5-ethynyl-2’-deoxyuridine(EdU)and PI can accurately distinguish cell cycle phases.And various immune cell subpopulations can be analyzed without cell sorting by combining surface marker staining with PI and Ki-67 staining.Here we review factors affecting cell cycle identification using the PI staining method and provide a standard operating protocol for the experiment.We established the method to combine EdU with PI for cell cycle detection and analysis of immune cell subpopulations,thus expanding the approaches for cell cycle detection.

关 键 词：流式细胞术 细胞周期 碘化丙啶 免疫细胞亚群 

分 类 号：Q34[生物学—遗传学]