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

MethodsX
Stephen T WardStuart M Curbishley

Abstract

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 sup...Continue Reading

References

Jul 5, 2006·The Journal of Experimental Medicine·Weihong LiuJeffrey A Bluestone
Jan 26, 2011·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Mario Roederer
Dec 14, 2011·European Journal of Immunology·Alicia N McMurchy, Megan K Levings
Mar 1, 2012·Journal of Immunological Methods·Benjamin J C Quah, Christopher R Parish
Aug 21, 2013·Clinical Immunology : the Official Journal of the Clinical Immunology Society·David C SolerKevin D Cooper

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Methods Mentioned

BETA
biopsies
FCS
flow cytometry

Software Mentioned

LightCycler
Flowjo

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