Annexin V-FITC/DAPI细胞凋亡检测法  

Annexin V-FITC/DAPI cell apoptosis detection method

作　　者：刘钰妮 王世恩 汪湘 任雪 夏燕 孙鹏[3] 曹春雨[1] LIU Yu-ni;WANG Shi-en;WANG Xiang;REN Xue;XIA Yan;SUN Peng;CAO Chun-yu(Three Gorges University School of Medicine,Hubei Provincial Key Laboratory of Tumor Microenvironment and Immunotherapy,Yichang 443002;Affiliated Hospital of Hubei University for Nationalities Hubei Provincial Key Laboratory of Occurrence and Intervention of Rheumatic Diseases,Enshi 445000;Hubei Sanxia Vocational and Technical College,Yichang 443002,China)

机构地区：[1]三峡大学医学院,肿瘤微环境与免疫治疗湖北省重点实验室,湖北宜昌443002 [2]湖北民族大学附属民大医院,风湿性疾病发生与干预湖北省重点实验室,湖北恩施445000 [3]湖北三峡职业技术学院,湖北宜昌443002

出　　处：《生物技术》2020年第4期352-361,共10页Biotechnology

基　　金：国家自然科学基金资助项目(81772833)。

摘　　要：[目的]建立新的荧光染料DAPI与FITC标记Annexin V联合的细胞凋亡流式细胞术检测方法,以用于具有橙红色荧光的药物处理细胞的流式细胞术凋亡检测。[方法]以倒置荧光显微镜成像和流式细胞仪分别检测不同浓度DAPI对活和死细胞的标记作用。以荧光酶标仪检测不同浓度DAPI处理细胞的荧光信号,并进行相关性分析。以流式细胞术比较Annexin V-FITC/DAPI双染法与Annexin V-FITC/PI双染法对无色和含红/橙色荧光药物导致的细胞凋亡。[结果]DAPI标记可用于区分死细胞和活细胞,DAPI标记死细胞的荧光信号和DAPI的浓度呈线性正相关。Annexin V-FITC/DAPI双染法与Annexin V-FITC/PI双染法相比,二者对不含红橙色荧光药物诱导的多种肿瘤细胞的凋亡检测结果无显著差异。与Annexin V-FITC/PI双染法相比,Annexin V-FITC/DAPI双染法可有效避免药物自身荧光与PI通道重合导致的流式检测干扰。[结论]成功建立了新的Annexin V-FITC/DAPI双染法用于细胞凋亡的流式细胞术检测,该方法能够避免具有红、橙色荧光基团的药物对的干扰检测凋亡。[Objective] To establish a novel flow cytometry detection method for the combination of fluorescent dye DAPI and FITC labeled Annexin V apoptosis detection by flow cytometry in cells treated with drugs with orange-red fluorescence.[Method] Inverted fluorescence microscope imaging and flow cytometry were used to detect the labeling effects of different concentrations of DAPI on live and dead cells. Fluorescence microplate reader was used to detect the fluorescence signal of cells treated with different concentrations of DAPI,and to conduct correlation analysis. Flow cytometry was used to compare Annexin V-FITC/DAPI and Annexin V-FITC/PI to colorless and red/orange fluorescent drugs-induced apoptosis.[Result]DAPI labeling could be used to distinguish between dead and live cells. The fluorescence signal of DAPI labeling dead cells was linearly correlated with the concentration of DAPI. Compared with Annexin V-FITC/PI double staining method,Annexin V-FITC/DAPI double staining method had no significant difference in the detection results of apoptosis of various tumor cells induced by red and orange fluorescent drugs. Compared with Annexin V-FITC/PI double staining method,Annexin V-FITC/DAPI double staining method could effectively avoid the interference of flow detection caused by the coincidence of drug autofluorescence and PI channel.[Conclusion]The new Annexin V-FITC/DAPI double staining method is successfully established for the flow cytometry detection of apoptosis. This method can avoid the interference of the drug pair with red and orange fluorescent groups to detect apoptosis.

关 键 词：Annexin V-FITC PI DAPI 流式细胞术 细胞凋亡检测 

分 类 号：R34[医药卫生—基础医学]