I have tried to enrich for T-regulatory cells from Lymph node and spleen of B6 mice and could not get more than 40-50% purity for CD4+ population. Have you tried the kit with >80% purity. What mice strain and which organ and if possible can you share the protocol and tricks?
Note for technical experts from Miltenyi: please refrain from commenting, I am seeking scientists advise. Thanks :)
That's weird. I've always had pretty good luck with the Miltenyi kits.
Yes, spleen is what you want to use.
The only thing I can think of... since the first step is a negative selection is that your cell to antibody ratio is off. So you are not getting rid of all the non-CD4 cells.
The other possibilty is your flow protocol for the analysis is somehow gated incorrectly. Even without separation you should get about 40% CD4 cells in the CD3 population.
Hopefully someone else has other ideas.
Thanks Shen for your reply.
" is that your cell to antibody ratio is off " I do not think so. I tried to play with the amount of antibody I am adding and the purity did not change.
" is your flow protocol for the analysis is somehow gated incorrectly. "
I gate out dead cells using the viability staining the gate on live population I gate on cells that are expressing CD4 and TCRb. I set my gating based on CD4-FMO and TCRb-FMO.
Do you use mannual seperation or AutoMACS?
Do you mind telling me what mice strain do you use?
My mouse work were almost completely on tuberuclosis and the vaccine/adjuvant field. So, the mouse strain that I used were C57BL/6 and Balb/c. I'm assuming that your mice are pretreated with something to enrich for T regulatory cells? Usually, I only isolate T cell populatons or other immune cells in mice for adopative transfer experiments or very small functional assays. Otherwise, I just use the human PBMCs. There is just not enough cells in mice for in vitro culture studies.
I always did the manual separation using the larger columns... I think it's the LX, but I can't remember.
Here's a stupid question, and I only ask this because I tend troubleshoot from the simplest things first. Did you stain for CD4 immediately after the isolation? And why did you use TCRb instead of CD3?
Here's something to try... It's a bit backwards... and this is only to see if it's the isolation or something else. If you have the CD4 positive selection kit, use it on the population after the CD25 selection. If you have a LOT of cells flow through, than you know that your negative selection for some reason is not purifying the CD4 cells enough. Usually positive selection is never a problem, it's always the negative selection that is the issue. But if you don't... you know that it's the analysis.
Another thing to try is to see what are the contiminated cells? In your FSC and SSC, what does this plot look like after you gate on the viable cells? Do you have a nice lymphocyte cluster or do you have other populations mixed in there? Did you try to stain for other markers to see what types of cells are making up the other 50% of the population?
Hope this helps. And hopefully someone else some other ideas.
For human Treg, I'm using Dynabeads™ Regulatory CD4+/CD25+ T Cell Kit (Thermofisher) that is a positive selection. This kit has given a good purity concerning Treg fraction but also concerning conventionnal CD4 T cells (CD25-) that are untouched fraction.
They have one for mouse Tregs (Dynabeads™ FlowComp™ Mouse CD4+CD25+ Treg Cells Kit). Maybe you can try it.
They say that cells could be put in culture after isolation.