title: Brefeldin A, but not monensin, enables flow cytometric detection of interleukin-4 within peripheral T cells responding to ex vivo stimulation with Chlamydia trachomatis.
abstract: Intracellular cytokine staining (ICS) assay optimization should include selection of suitable cytokine secretion inhibitors. Here, peripheral blood mononuclear cells (PBMC) from women with proven history of C. trachomatis genital tract infection were used to compare the ability of brefeldin A (BFA) and monensin (MN) to concurrently trap interferon-γ (IFN-γ), tumor necrosis factor (TNF), interleukin (IL)-4, and IL-17 within T cells responding to ex vivo stimulation with chlamydial antigen. While flow cytometric analyses showed similar intracellular levels of TNF, IFN-γ, and IL-17 among T cells treated with BFA or both BFA and MN, markedly more IL-4 was found inside T cells treated with BFA compared to those that received MN or BFA and MN. The latter findings oppose current ICS recommendations informing that ICS results are unaffected by concomitant use of BFA and MN, and also suggests that MN may be an unsuitable cytokine secretion inhibitor for ICS assays designed to measure intracellular IL-4 accumulation.
authors: Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA. rdv4@pitt.edu
authors: Rodolfo D
authors: RD
authors: Vicetti Miguel
authors: Samantha A
authors: SA
authors: Maryak
authors: Thomas L
authors: TL
authors: Cherpes
mesh_terms: D000704:Analysis of Variance; D000942:Antigens, Bacterial / Q000276:immunology; D020126:Brefeldin A / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D002690:Chlamydia Infections / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D020381:Interleukin-17 / Q000276:immunology / Q000378:metabolism; D015847:Interleukin-4 / Q000276:immunology* / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D020381:Interleukin-17; D014409:Tumor Necrosis Factor-alpha; D020126:Brefeldin A; D015847:Interleukin-4; D007371:Interferon-gamma; D008985:Monensin