流式细胞术检测细胞毒方法的建立

Establishment of flow cytometry assay of determination of cytotoxicity

目的 :建立一种用流式细胞仪测定细胞介导细胞毒活性的方法。方法 :用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记靶细胞 ,再用碘化丙碇 (PI)标记DNA筛选细胞膜已破坏的靶细胞。效靶比为 10 0∶1～ 6 2 5∶1,共孵育时间为 1～ 6小时 ,流式细胞仪收集 10 0 0个靶细胞并测定被杀细胞的百分率。结果 :CFSE是合适的标记靶细胞的荧光染料 ,18小时后也能很好地区分效靶细胞 ;靶细胞的自然死亡率低于 5 % ;杀伤百分率随着效靶比和共孵育时间的增加而增加。最佳效靶比为5 0∶1～ 2 5∶1,共孵育时间为 2～ 4小时。结论 :CFSE PI双荧光染料标记的流式细胞分析检测细胞毒具有多个优点 :避免放射性试剂的应用 ,敏感性增强 ,单细胞水平的分析。

Objective:To establish a new method of measuring cell-mediated cytotoxicity by flow-cytometric assay.Methods:The test principle is based on target cell labeling with CFSE and subsequent DNA-labelling with PI for identification of target cells with compromised cell membranes.Effectors and targets were incubated at E/T ratios from 100∶1 to 6 25∶1 at different time points from 1 h to 6 h,collect 1 000 target events and analyze specific cytotoxicity.Results:CFSE was found to be a suitable fluorochrome for labelling targets cells since it showed a good separation between effectors and target cells;the proportion of spontaneously dead target cell is below 5%;the cytotoxic activity showed a significant and positive correlation with E/T ratio and incubation period;the optimal E/T ratio and incubation period is 50∶1～25∶1 and 2 h～4 h.Conclusion:The benefits of flow cytometry to measure cytotoxicity are as follows:avoid radioactive substance;increase sensitivity at early timepoint;analyse at cell-to-cell basis;long-term incubation period is allowed.