title: Flow cytometric analysis of intracellular IFN-gamma, IL-4 and IL-10 in CD3(+)4(+) T-cells from rat spleen.
abstract: The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples, although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma, IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems.
authors.affiliation: Ottawa Hospital Research Institute, University of Ottawa, Ottawa Hospital - General Campus, Lab N1, 501 Smyth Road, Ontario K1H 8L6, Ottawa, Canada.
authors.forename: E M
authors.initials: EM
authors.lastname: Caraher
authors.forename: M
authors.initials: M
authors.lastname: Parenteau
authors.forename: H
authors.initials: H
authors.lastname: Gruber
authors.forename: F W
authors.initials: FW
authors.lastname: Scott
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology*; D005434:Flow Cytometry; D007371:Interferon-gamma / Q000096:biosynthesis*; D016753:Interleukin-10 / Q000096:biosynthesis*; D015847:Interleukin-4 / Q000096:biosynthesis*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D008985:Monensin / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011913:Rats, Inbred BB; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D017252:CD3 Complex; D007476:Ionophores; D011500:Protein Synthesis Inhibitors; D016753:Interleukin-10; D020126:Brefeldin A; D015847:Interleukin-4; D015759:Ionomycin; D007371:Interferon-gamma; D008985:Monensin; D013755:Tetradecanoylphorbol Acetate