Intracellular IL-10 detection in T cells by flowcytometry: the use of protein transport inhibitors revisited

Journal of Immunological Methods
Anne-Hilde MurisMariëlle Thewissen

Abstract

In the past two decades, interleukin-10 (IL-10) has gained much attention as an important regulatory cytokine involved in self-tolerance. Functional assessment of IL-10 producing immune cells is traditionally done by stimulation and measurement of cytokine production by flowcytometry. Thereby a protein transport inhibitor like monensin is used to accumulate the cytokine of interest intracellularly. In this study we elaborated on the monensin effect on cytokine detection and focused on IL-10 detection in human T cells. Peripheral blood mononuclear cells (PBMC) of 32 study subjects were isolated and stimulated with PMA/ionomycin, in the absence and presence of monensin, and stained intracellularly for IFN-γ, IL-4, IL-10 and IL-17A. Our results re-established that detection of IFN-γ+ and IL-4+ T cells benefited from the presence of monensin during stimulation. However, stimulation in the presence of monensin yielded lower proportions of IL-10+ T cells (0.45% (0.28-0.80) versus 0.80% (0.50-1.50) of CD4+ T cells, p<0.01), although monensin addition did result in an increased MFI (2431 (1273-4959) versus 1928 (1147-3760), p<0.01). Detectable fractions of IL-17A+ CD4+ T cells were not affected by monensin. A shorter incubation time, b...Continue Reading

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Citations

Mar 2, 2016·Journal of Crohn's & Colitis·Anna CarrascoFernando Fernández-Bañares
May 5, 2017·The Journal of Immunology : Official Journal of the American Association of Immunologists·Matthew R OlsonMark H Kaplan
Jun 19, 2019·The Journal of Clinical Investigation·Yoshiyuki MishimaR Balfour Sartor
Dec 17, 2020·European Journal of Neurology : the Official Journal of the European Federation of Neurological Societies·Max MimpenJan Damoiseaux
Jun 12, 2021·Advanced Science·Chao LiuGuozhen Liu

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