基于CD_(107a)标记流式细胞仪检测NK细胞毒性方法的建立

目的建立基于CD107a标记、利用流式细胞仪检测NK细胞毒性的方法。方法首先将外周血单个核细胞(PBM-Cs)与K562细胞以3∶1比例混合,2 h后加入莫能菌素,1.5 h后加入CD107a和CD56标记抗体,流式细胞仪分析CD107a阳性细胞的频率。其次观察CD107a抗体孵育时间、不同效靶比例对CD107a阳性细胞检测的影响。最后观察该方法与传统LDH释放法检测NK细胞毒活性的一致性。结果 NK细胞活化后可在其表面检测到高水平表达的CD107a分子,效靶细胞孵育结束后加入CD107a抗体可降低检测背景,低效靶比例同样具有检测敏感性。该方法与LDH释放法的检测结果一致。结论利用CD107a抗体标记检测NK细胞毒活性的方法具有快速、敏感、所需效应细胞少的优点。

Detection of NK cell cytotoxicity with CD107a antibody labeling by Flow Cytometry

Objective To detect NK cell cytotoxicity by flow cytometry following CD107a antibody labeling.Methods At first peripheral blood mononuclear cells(PBMC) were isolated and incubated with K562 target cells at a ratio of 3∶1.Two hours later,monensin was added into the culture system.After 1.5 hours CD107a and CD56 double positive cells were labeled with their corresponding antibodies,and analyzed by flow cytometry.The effects on detecting sensitivity by CD107a antibody in cubation time and the ratios of PBMC and K562 cells were also examined.Lastly we compared this method with the conventional LDH releasing method.Results CD107a positive cells could be easily detected on NK cells following stimulation.If CD107a antibody was added into culture medium after PBMC and K562 cells incubation,non-specific labeling was significantly decreased.The detecting result by this method was in conformity confirmative to that by the LDH releasing method.Conclusions The method we developed here for detecting cytotoxicity of NK cells was rapid,sensitive and needed a small amount of effectors.