title: Intracellular staining and detection of cytokines by fluorescence-activated flow cytometry.
abstract: The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining.
authors.affiliation: Department of Translational Medicine, Retrovirus Center, University of Pisa, Via del Brennero 2, I-56127, Pisa, Italy, giulia.freer@med.unipi.it.
authors.forename: Giulia
authors.initials: G
authors.lastname: Freer
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D018106:CD28 Antigens / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D039441:Integrin alpha4 / Q000235:genetics / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects*; D061251:Primary Cell Culture; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016707:Tissue Fixation
chemical_list: D000906:Antibodies; D018106:CD28 Antigens; D039441:Integrin alpha4; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate