Regulatory T cell isolation

5 posts / 0 new
Last post
N.arandi
N.arandi's picture
Regulatory T cell isolation

I want to isolate both regulatory T cells (Treg) and effector T cells for functional assay simultaneously by one kit but I can't find a suitable kit. I would be pleased if anyone can help me.
Thanks

Invitrogen Dyna...
Invitrogen Dynal Tech Support's picture
Hi

Hi

We have great Treg isolation kits for both human and mouse samples. The isolation process consists of two steps:
1. Negative isolation of CD4+ cells from PBMC
2. Positive isolation of CD4+CD25+ cells from the CD4+ population. These cells are then detached from the beads, giving you both the CD4+CD25+ Treg population and the CD4+CD25- Effector population. Both populations will be bead free and in perfect shape for expansion, supression and other cellular or molecular studies.

One great advantage with using the Dynabeads kit for the isolation is that the Treg cells have a high FOXP3 expression, which is a true indication of the regulatory properties of the cells.
The method and key benefits is more closely explained here:
http://tools.invitrogen.com/content/sfs/posters/Treg%20poster_web.pdf

We also have Dynabeads for physiological activation and/or expansion of Treg cells:
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-and-Tissue-Analysis/Cell-Isolation-and-Expansion/Cell-Expansion/Human-T-Cell-Expansion.html

I hope you find this useful!

Kristina

N.arandi
N.arandi's picture
Dear. Mrs. Kristina,

Dear. Mrs. Kristina,
Hi.
Thanks for your kindly help.
I have found such kit you mentioned but the major problem is that I believed that the CD4+CD25+  Treg siolated  from this kit have some contamination with effector T cells as we know that conventional CD4+ T cells express CD25 after their activation, so the isolated CD4+CD25+ T cells known as Treg are not really pure Treg and therefore are not suitable for further analysis for example functional assay. I have found the same kit from Miltenyi Biotech company and also another Treg isolation kit named CD4+CD25+CD127dim/- which unfortunately isolated only pure Treg and miss most of the CD4+CD127+ effector T cells.
I will be happy to hear from you If you have any comment on this issue.
Sincerely yours
Nargess Arandi

Invitrogen Dyna...
Invitrogen Dynal Tech Support's picture
Hi Nargess

Hi Nargess

Thank you for explaining in more detail what you are looking for.

In case anyone is confused about Treg characterisation, here is a short summary:
Treg cells are CD4+CD25+ and FOXP3+. However, activated T cells also have a transient expression of CD25 (caracterized as CD25low or CD25dim) and express CD127. Thus it is usual to characterize the Treg cells by their lack of CD127 expression as well as high FOXP3 and CD25 expression:

The aim then is to isolate cells which are CD4+ CD25(high)+ FOXP3+ CD127-.

When isolating Treg cells, it is possible to add anti-CD127 in the depletion step to remove any activated cells. This will give a high purity of Treg cells, but it will be at the cost of loosing many CD4 cells from the effector population (like you have experienced). So when you need both the effector and Treg populations this is not a good idea.

The Dynabeads Human Treg isolatin kit only isolates the CD25high population, not the CD25dim effector population. So, although it does not directly differentiate on the CD127 expression, the final Treg population, as you can see from the attached FACS resuls, will contain only a minute amount of CD127+ cells.
FACS: Treg population has been stained for FOXP3 and CD127, the purity of the Treg cells is 95%, which is very high. Of the CD4+CD25+ cells, 84% are FOXP3+ and CD127- and the isolated Treg cells have been shown to give 96% suppression.

I just though it would be nice to give you all these details, but I will of course leave it up to you to decide whether this purity will be sufficient for your application.

It would be possible to do a CD127 depletion on the Treg population after the isolation, but as this will cause loss of cells, it may not be advisable.

Please let me know if you have any questions or comments. Best wishes and good luck with your isolations!

Kristina

N.arandi
N.arandi's picture
Dear Kristina,

Dear Kristina,
Hi.
Thanks for your valuble and complete information.
I think I can use both Treg isolation kit named CD4+CD25+CD127dim/- for only Treg isolation and CD4+CD25+ kit for effector T cell isolation.
I really appreciate i you say your comment on this issue.
Thanks
Sincerely yours
Nargess Arandi