title: Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression.
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.
authors.affiliation: Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, Maryland 20892, USA. pchattop@mail.nih.gov
authors.forename: Pratip K
authors.initials: PK
authors.lastname: Chattopadhyay
authors.forename: Joanne
authors.initials: J
authors.lastname: Yu
authors.forename: Mario
authors.initials: M
authors.lastname: Roederer
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D023201:CD40 Ligand / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D008213:Lymphocyte Activation; D051379:Mice; D008985:Monensin / Q000032:analysis
chemical_list: D023201:CD40 Ligand; D008985:Monensin