A protocol for combining proliferation, tetramer staining and intracellular cytokine detection for the flow-cytometric analysis of antigen specific T-cells.
Flow-cytometry can be used in different ways in order to analyze or enumerate antigen specific T-cells. The three basic principles are direct staining of the T-cell receptor using so called tetramer reagents, staining intracellular cytokines following antigen-specific ex vivo T-cell activation or staining with dyes that are incorporated (increase in staining) or distributed between daughter cells (decrease in staining) upon proliferation in response to a specific antigen challenge. Each system has its advantages and disadvantages. Here we demonstrate that tetramer staining, cytokine flow cytometry and staining with CFDA-SE can be combined permitting the analysis of proliferation and cytokine production with a subset of T-cells specific for a single peptide antigen.
Tesfa
L
L
Institut für Medizinische Immunologie, Berliner Hochschulmedizin Charite, Charité Campus Mitte, Berlin, Germany. lidia.tesfa@charite.de
Volk
H D
HD
Kern
F
F
D000941:Antigens / Q000096:biosynthesis / Q000737:chemistry; D002455:Cell Division; D016207:Cytokines / Q000096:biosynthesis* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000166:cytology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000941:Antigens; D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma