Lecoeur H, Ledru E, Gougeon M L
Unité d'Oncologie Virale, Département SIDA et Rétrovirus, Institut Pasteur, Paris, France.
J Immunol Methods. 1998 Aug 1;217(1-2):11-26. doi: 10.1016/s0022-1759(98)00060-x.
The aim of this study was to define a simple and reliable method to detect simultaneously surface and intracellular antigens in apoptotic peripheral human lymphocytes. This approach requires a permeabilizing procedure for intracellular access of mAbs, which raises the important question of the influence of this procedure on parameters which identify apoptotic cells and on the surface expression of antigens. We compared the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantification of apoptotic lymphocytes, defined according to FSC/SSC criteria or following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface antigens with intracellular molecules, including Bcl-2 and cytokines (IFNgamma, TNFalpha, IL-2) was also analysed in the context of these three permeabilizing procedures. All the experiments were performed on PBMC from HIV-infected donors, known to undergo excessive apoptosis following short-term culture. We report that permeabilization with saponin is the only procedure which allows: (1) the preservation of lymphocyte morphology determined by the FSC/SSC parameters; (2) the quantification of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capacity (ABC); (4) the proper detection of intracellular membrane bound antigens (Bcl-2) and intracellular cytokines (IFNgamma, TNFalpha, IL-2); (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate that the simultaneous analysis of extracellular and intracellular antigens in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order.
本研究的目的是确定一种简单可靠的方法，用于同时检测凋亡外周血人淋巴细胞中的表面抗原和细胞内抗原。该方法需要一种通透处理，以使单克隆抗体能够进入细胞内，这就引发了一个重要问题，即该处理对识别凋亡细胞的参数以及抗原表面表达的影响。我们比较了三种目前常用的通透方法（0.05%皂树皂苷、0.1% Triton X - 100、70%乙醇）对根据FSC/SSC标准或7 - AAD染色定义的凋亡淋巴细胞定量的影响，以及对表面CD3、CD4、CD8、Fas、CD45R0分子检测的影响。在这三种通透处理的背景下，还分析了这些表面抗原与细胞内分子（包括Bcl - 2和细胞因子（IFNγ、TNFα、IL - 2））的联合检测。所有实验均在来自HIV感染供体的外周血单个核细胞（PBMC）上进行，已知这些供体的细胞在短期培养后会发生过度凋亡。我们报告称，皂树皂苷通透处理是唯一能够实现以下几点的方法：（1）通过FSC/SSC参数确定的淋巴细胞形态得以保留；（2）7 - AAD染色后凋亡淋巴细胞的定量；（3）可靠的表面免疫表型分析，保持良好的抗体结合能力（ABC）；（4）细胞内膜结合抗原（Bcl - 2）和细胞内细胞因子（IFNγ、TNFα、IL - 2）的正确检测；（5）凋亡细胞核、表面抗原和细胞内分子的联合检测。总之，这些观察结果表明，只要用于细胞通透的去污剂合适且后续染色程序按规定顺序进行，就可以轻松克服对属于复杂淋巴细胞群体（如PBMC）的凋亡细胞中的细胞外和细胞内抗原进行同时分析的问题。
