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Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Stellan Sandler)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.ORCID iD: 0000-0002-6771-7757
2019 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 144, article id e58848Article in journal (Refereed) Published
Abstract [en]

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Place, publisher, year, edition, pages
JOURNAL OF VISUALIZED EXPERIMENTS , 2019. no 144, article id e58848
Keywords [en]
Immunology and Infection, Issue 144, Single cell preparation, regulatory T cell, flow cytometry, Foxp3, Helios, Neuropilin 1
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-382478DOI: 10.3791/58848ISI: 000462905000030PubMedID: 30882793OAI: oai:DiVA.org:uu-382478DiVA, id: diva2:1307824
Funder
Swedish Research CouncilEXODIAB - Excellence of Diabetes Research in SwedenSwedish Diabetes AssociationSwedish Child Diabetes FoundationAvailable from: 2019-04-29 Created: 2019-04-29 Last updated: 2019-04-29Bibliographically approved

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Luo, ZhengkangThorvaldson, LinaBlixt, MartinSingh, Kailash

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