流式细胞技术和CCK-8法检测γδ T细胞活性的比较
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摘要
目的:比较流式细胞技术和CCK-8法检测γδ T细胞的活性。方法:密度梯度离心法分离外周血中单个核细胞(PBMCs),经磷酸抗原IPP刺激扩增培养14天,收集γδ T细胞。将收获的γδ T细胞分为两组:一组将乳腺癌MCF-7细胞与γδ T细胞共培养4 h,Calcein-AM/PI双染后流式细胞仪检测;另一组将乳腺癌MCF-7细胞与γδ T细胞共培养4 h,采用CCK-8试剂盒检测。比较两种方法检测γδ T细胞对MCF-7细胞毒活性的差别。结果:效靶比为5:1、10:1、20:1两种方法检测细胞毒活性结果分别为:流式细胞术组为37.30%±0.14%、40.30%±0.11%、50.70%±0.19%;CCK-8法组为17.90%±1.14%、25.70%±1.99%、43.10%±1.42%,两组之间差异有统计学意义(P<0.05)。Calcein-AM/PI双染后经流式检测法能较好的区分活细胞和凋亡细胞,且其结果的准确性、稳定性和重复性强于CCK-8法。结论:流式细胞术较CCK-8法稳定性好,准确性高,是一种优于CCK-8法的检测免疫细胞毒活性的方法。
Objective: To compare the activity of γδ T cells by flow cytometry and CCK-8.Methods: Mononuclear cells( PBMCs) were isolated from peripheral blood by density gradient centrifugation,and cultured for 14 days after stimulation with phosphoric acid antigen IPP.The γδ T cells were divided into two groups: MCF-7 cells were co-cultured with γδ T cells for 4 h with the methods of Calcein-AM/PI double staining.MCF-7 cells were incubated with γδ T cells for 4 h and detected by CCK-8.Comparison of the two methods in the detection of γδ T cells on MCF-7 cytotoxicity differences.Results: The results were as follows: Flow cytometry group: 37.3% ± 0.14%,40.3% ±0.11%,50.7% ± 0.19%,CCK-8 group: 17.90% ± 1.14%,25.70% ± 1.99%,43.10% ± 1.42%,the results were different( P < 0.05).Calcein-AM/PI double-staining flow detection method can better distinguish between living cells and apoptotic cells,and the accuracy,stability and repeatability of the results are stronger than CCK-8 method.Conclusion: Flow cytometry is a better and more accurate method than CCK-8 in the detection of immunocytotoxic activity.
Objective: To compare the activity of γδ T cells by flow cytometry and CCK-8.Methods: Mononuclear cells( PBMCs) were isolated from peripheral blood by density gradient centrifugation,and cultured for 14 days after stimulation with phosphoric acid antigen IPP.The γδ T cells were divided into two groups: MCF-7 cells were co-cultured with γδ T cells for 4 h with the methods of Calcein-AM/PI double staining.MCF-7 cells were incubated with γδ T cells for 4 h and detected by CCK-8.Comparison of the two methods in the detection of γδ T cells on MCF-7 cytotoxicity differences.Results: The results were as follows: Flow cytometry group: 37.3% ± 0.14%,40.3% ±0.11%,50.7% ± 0.19%,CCK-8 group: 17.90% ± 1.14%,25.70% ± 1.99%,43.10% ± 1.42%,the results were different( P < 0.05).Calcein-AM/PI double-staining flow detection method can better distinguish between living cells and apoptotic cells,and the accuracy,stability and repeatability of the results are stronger than CCK-8 method.Conclusion: Flow cytometry is a better and more accurate method than CCK-8 in the detection of immunocytotoxic activity.
引文
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[3]Brandes M,Willimann K,Moser B.Professional antigen-presentationfunction by human gammadelta T cells[J].Science,2005,309(5732):264-268.
[4]Yanling Wu,Yanping Ding,Yoshimasa Tanaka,et al.γδT cells and their potential for immunotherapy[J].Int J Biol Sci,2014,2(10):119-135.
[5]Wan Wenting,Li Ning,Liu Jing,et al.Comparison of CCK-8 and MTT assays in the detection of prostate cancer PC3 cells[J].Lishizhen Medicine and Materia Medica Research,2010,21(12):3046-3048.[万文婷,李宁,刘静,等.CCK-8法与MTT法检测人前列腺癌PC3细胞活性的比较研究[J].时珍国医国药,2010,21(12):3046-3048.]
[6]Rao Xiurong,Cui Bojing,Fu Fan,et al.Methodology for detection ofγδT-cell cytotoxicity by Calcein-AM/PI double-labeling coupling with flow cytometry[J].Immunological Journal,2016,32(7):620-625.[饶秀茸,崔博婧,付凡,等.Calcein-AM/PI双染法结合流式细胞术检测γδT细胞毒性的方法[J].免疫学杂志,2016,32(7):620-625.]
[7]Pievani A,Borleri G,Pende D,et al.Dual-functional capability of CD3+CD56+CIK cells,a T-cell subset that acquires NK function and retains TCR-mediated specific cytotoxicity[J].Blood,2011,118(12):3301-3310.
[8]Wu Yanhong,Wang Huiru,Deng Zhenling,et al.Advances in research of tumor biological immunotherapy[J].Science&Technology Review,2014,32(26):27-36.[吴艳红,王慧茹,邓振领,等.肿瘤生物免疫治疗研究进展[J].科技导报,2014,32(26):27-36.]
[9]Wang Liping.Study on the development of tumor biological immunotherapy[J].J Chin Pract Diagn Ther,2017,31(2):105-110.[王丽萍.肿瘤免疫治疗现状及进展[J].中华实用诊断与治疗杂志,2017,31(2):105-110.]
[10]Lu Shan,Li Suning,Fan Hong.Present situation and development suggestions of tumor immunotherapy technology and product development[J].China Biotechnology,2017,37(1):104-110.[卢姗,李苏宁,范红.肿瘤免疫治疗技术与产品开发的现状与发展建议[J].中国生物工程杂志,2017,37(1):104-110.]
[11]Zou Chang,Zhao Pan,Zhou Wenbin,et al.An exploratory study on the amplification of tumor cell specificγδT cells and its anti-tumor activities[J].Modern Oncology,2017,25(10):1540-1543.[邹畅,赵盼,周文斌,等.肿瘤特异性γδT细胞扩增体系的建立及其抗肿瘤活性研究[J].现代肿瘤医学,2017,25(10):1540-1543.]
[12]Chang L,Gusewitch GA,Chritton DB,et al.Rapid flow cytometric assay for the assessment of natural killer cell activity[J].J Immunol Methods,1993,166(1):45-54.
[13]Brunner KT,Mauel J,Cerottini JC,et al.Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro inhibition by iso-antibody and by drugs[J].Immunology,1968,14(2):181-196.
[14]Mhatre S,Madkaikar M,Ghosh K,et al.Rapid flow cytometry based cytotoxicity assay for evaluation of NK cell function[J].Indian J Exp Biol,2014,52(10):983-988.
[15]Jang YY,Cho D,Kim SK,et al.An improved flow cytometrybased natural killer cytotoxicity assay involving calcein AM staining of effector cells[J].Ann Clin Lab Sci,2012,42(1):42-49.