Sumner H, Abraham D, Bou-Gharios G, Plater-Zyberk C, Olsen I
Cell Enzymology Unit, Kennedy Institute of Rheumatology, London, U.K.
J Immunol Methods. 1991 Feb 15;136(2):259-67. doi: 10.1016/0022-1759(91)90012-5.
We have employed a method for permeabilizing lymphocytes with the detergent saponin in order to detect an intracellular protein simultaneously with surface antigens by flow cytometry (FCM). Using monoclonal antibodies specific for the murine CD2 receptor and for the lysosomal enzyme, beta-glucuronidase (Gus), we found that the expression of both of these antigens increased markedly when T cells were activated. Two sensitive methods were used to show that FCM provided an accurate measure of the actual number of CD2 and Gus molecules present in the lymphocytes. Immunogold electron microscopy revealed the precise ultrastructural localization of these different components and corroborated the specificity of the multiple labelling procedure for the simultaneous detection of surface and intracellular antigens. We also developed a three-colour FCM technique which we used to examine the changes in Gus expression in the CD4 and CD8 T cell sub-sets during activation.
我们采用了一种用去污剂皂角苷使淋巴细胞通透化的方法，以便通过流式细胞术（FCM）同时检测细胞内蛋白和表面抗原。使用针对小鼠CD2受体和溶酶体酶β-葡萄糖醛酸酶（Gus）的单克隆抗体，我们发现当T细胞被激活时，这两种抗原的表达均显著增加。我们使用了两种灵敏的方法来表明FCM能够准确测量淋巴细胞中存在的CD2和Gus分子的实际数量。免疫金电子显微镜揭示了这些不同成分的精确超微结构定位，并证实了用于同时检测表面和细胞内抗原的多重标记程序的特异性。我们还开发了一种三色FCM技术，用于检测激活过程中CD4和CD8 T细胞亚群中Gus表达的变化。
