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A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells

The suppression assay is a commonly performed assay, measuring the ability of regulatory T cells (Treg) to suppress T cell proliferation. Most frequently, Treg are obtained from the peripheral blood or spleen. Lower yields are obtained by isolation from other tissues, rendering downstream suppressio...

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Detalles Bibliográficos
Autores principales: Ward, Stephen T., Li, Ka-Kit, Curbishley, Stuart M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340526/
https://www.ncbi.nlm.nih.gov/pubmed/25729646
http://dx.doi.org/10.1016/j.mex.2014.08.012
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author Ward, Stephen T.
Li, Ka-Kit
Curbishley, Stuart M.
author_facet Ward, Stephen T.
Li, Ka-Kit
Curbishley, Stuart M.
author_sort Ward, Stephen T.
collection PubMed
description The suppression assay is a commonly performed assay, measuring the ability of regulatory T cells (Treg) to suppress T cell proliferation. Most frequently, Treg are obtained from the peripheral blood or spleen. Lower yields are obtained by isolation from other tissues, rendering downstream suppression assays challenging to perform. Furthermore, the importance of suppressive subpopulations of Treg favours their isolation by fluorescent-activated cell sorting. Here we describe a method to isolate Treg from human tissues, using colorectal cancer tissue as an example. Treg suppressive capacity was further examined by expression of CCR5 to demonstrate the ability of our method to assess the suppressive capacity of regulatory T cell subsets. To optimise the standard suppression assay to achieve our research aims, the following modifications were made: • Treg, isolated from tissues, were sorted directly into a well-plate. • Responder T cells, which had been fluorescently-labelled prior to sorting, were added directly into the well-plate. • Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd., UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2. This method quantified the suppression of responder T cell proliferation by small numbers of strictly-defined Treg populations isolated from tissues.
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spelling pubmed-43405262015-02-25 A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells Ward, Stephen T. Li, Ka-Kit Curbishley, Stuart M. MethodsX Immunology and Microbiology The suppression assay is a commonly performed assay, measuring the ability of regulatory T cells (Treg) to suppress T cell proliferation. Most frequently, Treg are obtained from the peripheral blood or spleen. Lower yields are obtained by isolation from other tissues, rendering downstream suppression assays challenging to perform. Furthermore, the importance of suppressive subpopulations of Treg favours their isolation by fluorescent-activated cell sorting. Here we describe a method to isolate Treg from human tissues, using colorectal cancer tissue as an example. Treg suppressive capacity was further examined by expression of CCR5 to demonstrate the ability of our method to assess the suppressive capacity of regulatory T cell subsets. To optimise the standard suppression assay to achieve our research aims, the following modifications were made: • Treg, isolated from tissues, were sorted directly into a well-plate. • Responder T cells, which had been fluorescently-labelled prior to sorting, were added directly into the well-plate. • Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd., UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2. This method quantified the suppression of responder T cell proliferation by small numbers of strictly-defined Treg populations isolated from tissues. Elsevier 2014-08-28 /pmc/articles/PMC4340526/ /pubmed/25729646 http://dx.doi.org/10.1016/j.mex.2014.08.012 Text en © 2014 The Authors https://creativecommons.org/licenses/by/3.0/This work is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/) .
spellingShingle Immunology and Microbiology
Ward, Stephen T.
Li, Ka-Kit
Curbishley, Stuart M.
A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells
title A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells
title_full A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells
title_fullStr A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells
title_full_unstemmed A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells
title_short A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells
title_sort method for conducting suppression assays using small numbers of tissue-isolated regulatory t cells
topic Immunology and Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340526/
https://www.ncbi.nlm.nih.gov/pubmed/25729646
http://dx.doi.org/10.1016/j.mex.2014.08.012
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