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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2014
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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. |
format | Online Article Text |
id | pubmed-4340526 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
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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