流式细胞技术和CCK-8法检测γδ T细胞活性的比较

Comparison of the activity of γδ T cells by flow cytometry and CCK-8

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.