Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression
Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression
复制标题
DOI：
10.1038/nprot.2006.1
复制
发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Roederer, Mario
中科院分区：
文献类型：
--
作者：
Chattopadhyay, Pratip K.;Yu, Joanne;Roederer, Mario
该方案详细介绍了一种鉴定对抗原产生应答的CD4 + T细胞的方法。该方法依赖于对CD154的检测，CD154是一种共刺激细胞表面蛋白，在激活时由CD4 + T细胞表达，可用于纯化具有多种功能的活CD4 + T细胞。为了检测CD154，在6到24小时的刺激期间，将荧光标记的抗体与细胞样本、肽（或完整抗原）以及莫能菌素一起培养。（注意该检测方法与布雷菲德菌素A不兼容。）刺激后，细胞用任何其他感兴趣的抗体染色，然后通过流式细胞术进行分析或通过细胞分选进行纯化。与其他检测方法不同，该方法允许同时评估其他细胞表型或功能，与下游基于RNA的检测方法兼容，并能保持细胞活力。该方案可在9小时内完成。
This protocol details a method to identify CD4+ T cells that respond to antigens. The method relies on detection of CD154, a costimulatory cell surface protein that is expressed by CD4+ T cells upon activation, and can be used to purify live CD4+ T cells of diverse function. To detect CD154, fluorescently labeled antibodies are cultured with cell samples, peptides ( or whole antigens) and monensin during a 6- to 24-h stimulation period. ( Note that the assay is not compatible with brefeldin A.) After stimulation, cells are stained with any other antibodies of interest and then are analyzed by flow cytometry or purified by cell sorting. Unlike other assays, this method allows simultaneous assessment of other cell phenotypes or functions, is compatible with downstream RNA-based assays and preserves cell viability. This protocol can be completed in 9 h.