Mehta B A, Maino V C
Becton Dickinson Immunocytometry Systems, San Jose, CA 95131, USA.
J Immunol Methods. 1997 Oct 13;208(1):49-59. doi: 10.1016/s0022-1759(97)00127-0.
Assessment of T cell activation has traditionally been performed by measuring proliferation as a function of 3[H]-thymidine incorporation, or secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture. An alternative method for detection of proliferation at the single cell level utilizes incorporation of bromodeoxyuridine (BrdU), an analog of thymidine, into cellular DNA. After appropriate fixation and permeabilization of the cells, a monoclonal antibody (mAb) against BrdU conjugated with a fluorescent dye is employed to measure by flow cytometry the incorporated BrdU. Here, we report a flow cytometric procedure which can be used for the simultaneous detection of BrdU incorporation, activation markers such as CD69 and CD25, and intracellular cytokines in T cell subsets from activated PBMC. Our observations are consistent with the proposal that cytokine synthesis and cell proliferation occur sequentially in CD4+ T cells stimulated with the superantigen staphylococcal enterotoxin B (SEB). The majority of cells expressing the cytokines IFN-gamma and IL-2 at 48 h appear to have undergone DNA synthesis, however all proliferating cells do not express IFN-gamma or IL-2. The methods presented in this report offer a unique approach for studying simultaneous expression of key cellular activation events in phenotypically resolved lymphocyte populations.
传统上，T细胞活化的评估是通过测量作为3[H] - 胸腺嘧啶核苷掺入函数的增殖，或通过测量培养的活化外周血单核细胞（PBMC）分泌的细胞因子来进行的。一种在单细胞水平检测增殖的替代方法是利用胸腺嘧啶核苷类似物溴脱氧尿苷（BrdU）掺入细胞DNA中。在对细胞进行适当的固定和通透处理后，使用与荧光染料偶联的抗BrdU单克隆抗体（mAb）通过流式细胞术测量掺入的BrdU。在此，我们报告一种流式细胞术程序，该程序可用于同时检测活化PBMC中T细胞亚群的BrdU掺入、活化标志物如CD69和CD25以及细胞内细胞因子。我们的观察结果与以下提议一致，即在用超抗原葡萄球菌肠毒素B（SEB）刺激CD4 + T细胞时，细胞因子合成和细胞增殖是依次发生的。在48小时时表达细胞因子IFN-γ和IL-2的大多数细胞似乎已经经历了DNA合成，然而，所有增殖细胞并不都表达IFN-γ或IL-2。本报告中介绍的方法为研究表型解析的淋巴细胞群体中关键细胞活化事件的同时表达提供了一种独特的方法。
