Annexin V/PI法流式细胞术检测细胞凋亡的影响因素  

Factors influencing detection of apoptosis by flow cytometry using Annexin V/PI staining

作　　者：管强东 杨健[2] 刘艳青[1] 熊建平 钟义红[1] 沈链链[1] GUAN Qiang-dong;YANG Jian;LIU Yan-qing;XIONG Jian-ping;ZHONG Yi-hong;SHEN Lian-lian(School of Public Health,Nanjing Medical University,Nanjing 211166,China;Department of Urology,The Second Affiliated Hospital of Nanjing Medical University,Nanjing 210011,China)

机构地区：[1]南京医科大学公共卫生学院,江苏南京211166 [2]南京医科大学第二附属医院泌尿外科,江苏南京210011

出　　处：《生物技术》2022年第6期710-714,共5页Biotechnology

基　　金：南京医科大学科技发展基金项目(NMUB2019011)。

摘　　要：[目的]探讨消化液、细胞数量及试剂温度对Annexin V/PI法流式细胞术检测细胞凋亡率的影响。[方法]细胞经Accutase或无EDTA胰酶消化后比较检测结果;含有0.5×10^(5)、1×10^(5)、2×10^(5)细胞的样本按照说明书推荐(默认含有1×10^(5)细胞)加入试剂后,比较检测结果;细胞加入室温或4℃试剂后比较检测结果。以上比较均用未处理细胞和秋水仙素处理细胞分别进行。[结果]对于未处理细胞而言,与无EDTA胰酶相比,Accutase能显著减少凋亡率(2.70%vs 18.05%);而经药物处理的细胞,Accutase组凋亡率也有一定程度减少(22.32%vs 26.50%),但差异没有未处理细胞大。未处理细胞随着细胞数量的增加,PI信号并未发生明显改变,Annexin V-FITC信号与细胞数量成反比,统一十字门分析的凋亡率分别是:2.46%、1.85%、1.35%。对于药物处理的细胞差异尤其明显,统一十字门分析的凋亡率分别是:28.83%、21.27%、15.59%,因此不能用统一的十字门分析。加入4℃试剂的未处理细胞的凋亡率明显高于加入室温试剂的细胞的凋亡率(3.77%vs 8.13%),而对于药物处理的细胞,两者基本无差别(22.94%vs 22.18%)。[结论]用Annexin V/PI法流式细胞术检测细胞凋亡率时,使用Accutase消化液;样本严格计数,使细胞数符合说明书要求;使用室温试剂。[Objective]To investigate the effects of cell dissociation,numbers of cells contained in each sample and the temperature of the reagents on apoptotic rates detected by flow cytometry,when using Annexin V/PI staining.[Method]Cells were dissociated with Accutase or EDTA-free trypsin and apoptotic rates were compared.Samples containing 0.5×10^(5),1×10^(5) or 2×105cells were collected and the same reagents,which was suitable for 1×10^(5) cells according to the instruction of the kit,were added to each sample and apoptotic rates were compared.The reagents that have been equilibrated at room temperature or cold reagents were added and apoptotic rates were compared.All the comparisons were conducted both in untreated and drug-treated cells.[Result]For both untreated and treated cells,the apoptotic rates of the samples dissociated by Accutase were lower than those of the samples by EDTA-free trypsin,the difference of former being much more significant(2.70%vs 18.05%,22.32%vs 26.50%).For both untreated and drug-treated cells,the Annexin V-FITC fluorescence signal decreased as cell numbers increased,with the PI fluorescence signal generally unchanged.By placing the same quadrants,the proportion of apoptotic cells of the sample containing 0.5×10^(5),1×10^(5),2×10^(5) untreated cells was 2.46%,1.85%,1.35%,respectively.Similarly,the proportion of apoptotic cells of the sample containing 0.5×10^(5),1×10^(5),2×10^(5) drug-treated cells were 28.83%,21.27%,15.59%,respectively.Resultantly,the proportion of apoptotic cells of each sample can not be quantified by placing the same quadrants,especially for drug-treated cells.For untreated cells,the apoptotic rates of samples to which cold reagents were added were significantly higher than those of samples to which reagents that have been equilibrated at room temperature were added(3.77%vs 8.13%).For drug-treated cells,no significant differences were noted(22.94%vs 22.18%).[Conclusion]When using Annexin V/PI staining to detect apoptosis by flow cytometry,it is suggested that

关 键 词：流式细胞术 Annexin V/PI法 细胞调亡 影响因素 

分 类 号：R331[医药卫生—人体生理学]