四色法流式细胞术测定淋巴细胞内细胞因子的方法学探讨

Determination of Intracellular Cytokines in Human Lymphocytes by Four-color Flow Cytometry

目的 建立流式细胞术测定淋巴细胞内细胞因子的方法。方法 正常人外周血在PMA、iono-mycin和BFA存在的条件下在体外短期培养,进行细胞膜表面抗原和细胞内细胞因子染色,再用流式细胞仪对淋巴细胞内细胞因子进行测定,并对分析方法中的一些测定条件进行了探讨。结果 刺激剂的刺激时间和不同的固定穿透剂对结果有较大的影响,但在最佳条件下,该方法的实验内变异比较小,平均CV在8％左右;实验间的变异稍大,平均CV在20％左右。结论 ①流式细胞术是单细胞水平检测细胞内细胞因子的理想方法,联用IFN-γ/IL-4/CD8/CD3四色荧光标记,可以在一支检测管中同时了解T细胞亚群中两类细胞因子的表达情况;②细胞内细胞因子检测的影响因素较多,分析时需注意选择适当的测定条件。

Objective To set up the technique of detection of intracellular cytokines by four-color flow cytometry. Methods Cytokines in human T lymphocytes were assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure after stimulated with phorbol-12-myristate-13-acetate (PMA) and ion-omycm under the influence of Brefeldin (BFA). The effects of Multi-factor on method were explored. Results The time of stimi.lation and different fixation/permeabilization have great effects on the results,but in the optimal conditions,the in-tratest variation was low,the mean value was about 8%,the intertest variation was high a little,the mean value was about 20% Conclusion (1)Flow cytometry is a good tool to detect the intracellular cytokines at the single cell level. It is possible to measure the co-expression of two cytokines in T lymphocyte subsets in the detected tube by using four-color flow cytometry. (2)There are many influence factors in the method. It is noted to select the appropriate conditions at the analysis.