Journal Article10.1038/NPROT.2006.1
Live-cell assay to detect antigen-specific CD4 + T-cell responses by CD154 expression
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TL;DR: This protocol details a method to identify CD4+ T cells that respond to antigens that allows simultaneous assessment of other cell phenotypes or functions, is compatible with downstream RNA-based assays and preserves cell viability.
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Abstract: 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.
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TL;DR: A novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis, and CD107-expressing CD8+, expressing cognate T cell receptors (TCR), is presented.
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TL;DR: This article reviews recent developments in this field of research, with main emphasis on structure and expression of CD40 and its ligand; (2) CD40 signal transduction; (3) in vitro function ofCD40 on different cell types; and (4) in vivo functions of CD 40/CD40‐L interactions.
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TL;DR: It is suggested that CD40 is essential for T cell-dependent immunoglobulin class switching and germinal center formation, but not for in vivo Tcell-dependent IgM responses and T cell -independent antibody responses.
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Meenakshi Roy,Thomas J. Waldschmidt,Alejandro Aruffo,Jeffrey A. Ledbetter,Randolph J. Noelle +4 more
TL;DR: Differences in the expression of gp39 on activated naive and memory T cells were observed, as well as differences in requirements for optimal gp39 expression on these subsets, suggesting that density of gp40 on the activated T cells plays an important role in determining effector function.
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A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles.
TL;DR: This assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h, providing a means to purify viable antigen-specific CD4+ T cells for further analysis.
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