Schütz Christian, Fischer Karin, Völkl Simon, Hoves Sabine, Halbritter Dagmar, Mackensen Andreas, Fleck Martin
University of Regensburg, Department of Internal Medicine I, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany.
J Immunol Methods. 2009 May 31;344(2):98-108. doi: 10.1016/j.jim.2009.03.008. Epub 2009 Mar 28.
Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches.
新型免疫抑制策略旨在特异性清除自身免疫和同种异体移植排斥反应中导致器官损伤的T细胞。在此，我们介绍一种基于流式细胞术的新检测方法，该方法能够可靠且高效地检测以抗原特异性方式被清除的T细胞。一种利用两种膜染料PKH26和PKH67的稳定细胞标记技术已与膜联蛋白V和7-氨基放线菌素（7-AAD）染色相结合，以检测凋亡细胞。一种差异门控策略使我们能够独立确定每个PKH染色的T细胞亚群的活力/凋亡情况。能够同时分析不同抗原特异性的T细胞混合物中的凋亡情况，使该检测方法成为进一步开发新型抗原特异性免疫抑制方法的卓越工具。
