Determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocytes by flow cytometry following whole-blood culture.
This report describes a whole-blood flow cytometric method for the determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocyte subpopulations. Conjugated anti-cytokine antibodies and commercially available reagents for cell fixation and permeabilization were used. Canine peripheral blood was cultured with a combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to promote cytokine synthesis in each cell, along with monensin to increase the sensitivity of the method by retaining IFN-gamma and IL-4 within the cell to detectable levels. The optimum concentrations of PMA and ionomycin were determined. Maximum IFN-gamma expression from both CD4+ and CD8+ T lymphocytes was detected after 6 h of incubation of cell culture, while maximum IL-4 production took 6 h from CD4+ cells and 4 h from CD8+ cells. This method is a simple immunologic technique for measuring intracellular cytokines which could be of value in the investigation of canine immunological response mainly in various intracellular and extracellular infections, since IFN-gamma and IL-4 are considered key cytokines activating the cellular and humoral immunity, respectively.
Department of Veterinary Public Health, National School of Public Health, Athens, Greece. dermpap1@otenet.gr
Emmanouil I
EI
Papadogiannakis
Vasilios I
VI
Kontos
Maria
M
Tamamidou
Anastasia
A
Roumeliotou
D000818:Animals; D015704:CD4 Antigens / Q000096:biosynthesis / Q000097:blood; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D016827:CD8 Antigens / Q000096:biosynthesis / Q000097:blood; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D004285:Dogs / Q000097:blood / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000097:blood; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000097:blood; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D008985:Monensin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015704:CD4 Antigens; D016827:CD8 Antigens; D015847:Interleukin-4; D015759:Ionomycin; D007371:Interferon-gamma; D008985:Monensin; D013755:Tetradecanoylphorbol Acetate