Calcein-AM/PI双染法结合流式细胞术检测γδT细胞毒性的方法  

Methodology for detection of γδT-cell cytotoxicity by Calcein-AM/PI double-labeling coupling with flow cytometry

作　　者：饶秀茸 崔博婧 付凡 王宇环 马飞 

机构地区：[1]深圳市合一康生物科技股份有限公司,518000

出　　处：《免疫学杂志》2016年第7期620-625,共6页Immunological Journal

基　　金：深圳市战略新兴产业发展专项资金课题(CYZZ20130329145313934);深圳市生物产业发展专项资金(2014年第三批扶持计划;CXZZ20140509144527788)

摘　　要：目的建立和优化钙黄绿素-AM（Calcein-AM）和碘化丙啶（propidium iodide,PI）双染法结合流式细胞术检测γδT细胞细胞毒活性的方法。方法利用Calcein-AM标记靶细胞,然后与效应细胞共孵育24 h,结束后加入PI,最后流式细胞术检测;通过BD公司绝对细胞计数管验证上述流程的稳定性和可靠性。结果 Calcein-AM能在较低浓度（0.1～0.5μmol/L）下对靶细胞在宽密度范围（0.1～10）×10^6/ml内进行很好标记,而且24 h内对细胞活率无影响,同时还能与未标记细胞进行很好的区分;整个流程具有很好的稳定性和可靠性。结论建立并优化了Calcein-AM/PI双染法结合流式细胞术检测γδT细胞细胞毒效应的方法,本方法除操作简单、重复性好、灵敏度高外,还能更好的区分死亡细胞的来源。This study aimed to establish and optimize a Calcein-AM/PI double-labeling method coupling withflow cytometry for the detection of γδ T-cell cytotoxicity.The target cells were stained by Calcein-AM and thenco-cultured with γδ T-cells for 24 hours.After coculture,PI was added into the cultures and followed by flowcytometry.In the end,BD Tru COUNTTMTubes was used to verify the stability and reliability of the whole process.Datashowed that Calcein-AM（0.1-0.5 μmol/L） could stain the target cells in a wide range of density（0.1×10^6-10×10^6/ml）.Calcein-AM staining had no obvious effect on target cell activity and fluorescence intensity in 24 hours.Importantly,the whole process was stability and reliability.In conclusion,the improved cytotoxicity test has established byCalcein-AM/PI double-labeling method coupling with flow cytometry,which is a sample operation with goodrepeatability,and can be carry out at single cell level.

关 键 词：钙黄绿素-AM ΓΔT细胞 细胞毒活性 流式细胞术 

分 类 号：R392[医药卫生—免疫学]