I use FoxP3-GFP mice to sort Treg cells as GFP+. But once I plate my Tregs with CD3, CD28 and IL2 to maintain the survival of Treg cells and observe its effect in vitro, after 72h (normally due to small contamination of TCD4 cells), I only observe 30-40% Tregs, because TCD4 cells proliferate faster than Tregs.
How could I optimize the protocol in order to get only proliferation of Treg cells and a good purity in my culture? I also tried to isolate Tregs as CD4+ CD25+, quantity of Tregs cells was much better but not a good marker CD25+.
Thank you
CD25 is an activation marker, that you could find on different subpopulation of Th CD4+ and even CD8+ cells. Actually the identification of Treg is so far based on different markers among whom CD3, CD4, CD25, FoxP3. Under different points of view actually it's not easy to distinguish TCD4+ and Treg with FACS alone, and usually one could play with transcription factors, but I think you want to manipulate your cells as little as possible, and any treatment will probably lead to an inferior cell yield. I suggest you to sort the cells twice as GFP+, in order to reduce the contamination. Or you could try giving a calcium stimulus with PMI/Ionomicyn that will reactivate the cells that were already activated. CD4+ cells in normal mice may be not usually activated because of lack of particular stimuli, and so they may be CD25- . In this case you could use positive selection with CD25+ more efficiently.
What is your purity of FoxP3+ CD4 cells after FACS sorting?
CD3, CD28, and IL2 should be fine, but what concentrations are you using (are the antibodies platebound)? Do you replace/add more media within the 72 hours with new IL2?
Thanks for yours suggestions. PMA/Ionomicyn treatment would activate T CD4 also, becoming CD25+. So maybe trying to sorting again with GFP+
I get purity of 90-95% but even after that contamination of CD4 T cells are much greater in culture.
I would suggest to perhaps doing a magnetic bead sort on CD4 T cells (negative selection) prior to the FACS sort if you want to go that route. Work with polypropylene tubes to improve recovery, and if someone else is doing the FACS sorting just make sure they know you want purity above all else.
It shouldn't be a problem to get around 98-99% purity when you test stain a small portion of the purified cells for FoxP3 expression. Ensure that the cells are getting plenty of IL2 during the culture step. With just CD3, CD28, and IL2 supplementation you should have around 80% FoxP3+ CD4 T cells after 3 days.
you can use FACS sorting of CD4+CD25high and then test for Foxp3 presence by FACS or RT-PCR.
- Badges
- Science method