I am planning to sort Tregs from an HIV patient. Since in HIV CD25 might be up-regulated on conventional CD4 T cells, and Foxp3 can be transiently up-regulated in activated conventional CD4 T cells, I do not know what markers to use for the sorting.
I would like to have high purity of Tregs in the sorting, what do you recommend I use? I have heard about GARP, which has been suggested as a marker to discriminate true Tregs (Wang et al., PNAS, 2009).
What are you planning to do after you sort?
I ask because to sort Tregs, you should at least look for CD4+ CD25+ FoxP3+ cells, and to stain for FoxP3, you will need to permeabilize the cells.
If you're permeabilizing, perhaps looking at T-bet or GATA3 to discriminate Th1/Th2 cells from Tregs may be useful.
Hi, an alternative to flow sorting might be immunomagnetic isolation of CD4+CD25+ cells - a two step isolation strategy starting with depletion of all non-CD4+ cells followed by positive isolation of CD25+ cells. Finally, beads and Abs are removed from the cells and the cells are ready for any downstream application and can in addition be expanded using e.g. beads coated with anti-CD3 and anti-CD28 Abs. After this workflow the target cells (FoxP3+) have suppressor capacity and suppress proliferation of CD4+CD25- effector T-cells in the presence of CD3 activation.
kind regards
ketil winther pedersen
Hi, I need the Tregs afterwards to perform a proliferative assay, I want to test how is the proliferation of Tregs afected when adding a reagent. So I can not permeabilize the cells.
My fear is that in HIV CD25 might be upregulated in conventional Tcells so I will get a mixture of Tregs and Tconv cells after sorting with CD4 and CD25. So I was wondering if I could use alternative markers. Is CD127 a good option?
Thanks!
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