The Use of Flow Cytometry to Assess the State of Chromatin in T Cells.
The Use of Flow Cytometry to Assess the State of Chromatin in T Cells.
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DOI：
10.3791/53533
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发表时间：
2015-12-17
期刊：
影响因子：
--
通讯作者：
Rawlings JS
中科院分区：
文献类型：
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作者：
Bingham KN;Lee MD;Rawlings JS
在适当的免疫应答期间，静止的T细胞在抗原呈递至其抗原特异性T细胞受体时被激活。这导致仅那些携带识别抗原的受体的T细胞的克隆增殖。染色质去凝聚是T细胞活化的标志，并且是T细胞在抗原接合后获得增殖能力所必需的。这种染色质凝聚的变化可以用针对组蛋白的抗体检测到。这些抗体不能像在活化的T细胞中那样与幼稚T细胞中的表位结合。我们描述了如何同时染色T细胞特异性的表面标记，跟踪与可固定的死细胞染色的活力，并通过组蛋白H3蛋白的细胞内染色测量染色质状态。通过流式细胞术分析染色的细胞，并将染色质凝聚状态测量为组蛋白H3染色的平均荧光强度（MFI）。T细胞活化期间的染色质去浓缩被证明为MFI的增加。
During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in naïve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI