title: Detection of antigen-specific T cells based on intracellular cytokine staining using flow-cytometry.
abstract: CMV-specific T cells may be detected and quantified after antigen-specific stimulation based on the induction of cytokines as a readout system. Secreted cytokines may be detected from the supernatant of stimulated cells using ELISA. Alternatively, antigen-specific cytokine-secreting cells may be enumerated using an ELISPOT assay. These assays generally rely on the detection of IFNγ and do not allow for a simultaneous assessment of several cytokines on a single cell basis. Here we describe a flow-cytometry based method to analyze CMV-specific CD4 T cells after specific stimulation with a whole antigen lysate. In this assay, cytokine secretion from stimulated cells is blocked by the addition of brefeldin A. Using a panel of fluorescently labeled antibodies, not only intracellularly accumulated cytokines but also phenotypical characteristics of specifically activated T cells may be quantified in a multiparameter staining approach.
authors.affiliation: Department of Transplant and Infection Immunology, Saarland University, Homburg(Saar), Germany.
authors.forename: Tina
authors.initials: T
authors.lastname: Schmidt
authors.forename: Martina
authors.initials: M
authors.lastname: Sester
mesh_terms: D000941:Antigens / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*
chemical_list: D000941:Antigens; D016207:Cytokines