Hi all,
I am trying to isolate Tregs for further downstream analysis - in vitro stimulation and cytokine analysis. I have been using the Miltenyi Treg (CD4+CD25+) protocol which involves two steps: negative isolation of CD4+ cells and positive isolation of CD25+ cells. I have not been able to consistently achieve high purity and mostly get a purity of around 45%. Has anyone experienced similar and are there strategies to improve the purity using this Miltenyi kit? OR any other alternative methods to isolate Tregs with guaranteed high purity?
Thanks in advance.
Mildred
Hi Mildred,
This may not be a feasible answer but if its important to have a very pure population I would consider using the Foxp3-GFP mouse from Jax and isolate using sorting on GFP. If you can sort that is the way I would go. Not too spendy either. Alternatively you could add a CD127 negetive selection at the end (collect flow through), if you have enough starting material...
Example:
https://www.jax.org/strain/006769
https://www.jax.org/strain/023800
NM
Thanks Nathaniel. Sorry I should have mentioned that I am working with human samples.
Hi Mildred,
You can try EasySep™ Human CD4+CD25+ T Cell Isolation Kit. I think, it will increase purity.
Hi Mildred,
Are you still looking for a protocol for isolation of purified T regs? As Mahmudul mentioned, you can try our EasySep kits.
We have a kit which uses a similar protocol to what you are using with Miltenyi, i.e. negative isolation of CD4+ cells followed by positive selection of CD25+ cells (https://www.stemcell.com/products/complete-kit-for-human-cd4-cd25-t-cells.html), or kit for the isolation of CD4+CD127lowCD25+ cells (https://www.stemcell.com/products/easysep-human-cd4-cd127lowcd25-regulatory-t-cell-isolation-kit.html).
We also have few more options, depending on the exact population of cells that you are interested and the method of isolation (positive, negative). The kits are column-free, fast and reproducible.
I am the representative for STEMCELL Technologies for cell separation products in Oxford. So if you would like to try any of these kits, please get in touch ([email protected]) and I will arrange a sample for you.
All the best with your research!
Elwira
Here are some references:
Mathieu F. Chevalier et al. Phenotype Alterations in Regulatory T-Cell Subsets in Primary HIV Infection and Identification of Tr1-like Cells as the Main Interleukin 10-Producing CD4+ T Cells. J. Infect. Dis. 211(5):769-79 (March 2015);
Rajendra Pahwa et al. Isolation and expansion of human natural T regulatory cells for cellular therapy.J Immunol Methods (October 23, 2010);
