Andrew remember the Treg phenotype is CD4+CD25hi CD127lo. It comprises about 2-5% of peripheral blood. To do Treg sorting its best to use a flow cytometer. Beads will put out effector cells which is not Treg cells. Hope this helps

Around 2.5-4% of total CD4+ T cells

I think the yield is low as I used to get about 1million from 25 million CD4+ cells. Are you doing a Negative selection or a Positive selection?

I've been using the Miltenyi Treg isolation kit, which has two stages -first depletion of non-CD4 and CD127hi cells, followed by positive selection of CD4+CD25+CD127lo/neg cells, using MACS columns (not tried it on the autoMACS separator yet).

Andrew Conway Morris, we use the same kit. From 20 ml blood sample (perkoll obtaining of pbms preceding) the average outcome is about million cd4+cd25+cd127lo/- cells (column output). however this population is not often pure as determined by flow cytometer as we used mini macs instead of LD columns.

Hi Andrew,

we usually get 1-2% from total PBMC.

In our lab we use a different protocol for isolation of Treg. We positively enrich CD25high cells by applying limited amounts of CD25-Microbeads (Miltenyi) to the PBMC. The CD25+ cells are afterwerds depleted from contamination using CD8, CD14 and CD19 Dynabeads (Invitrogen).

We come out with nicely pure Treg, expressing high levels of Foxp3, and low CD127...

For further reading:

http://www.ncbi.nlm.nih.gov/pubmed/11390435

Greetings,

Mario

Hi,

I am wondering what is the aproximate purity of your "Treg fraction" (when using the Miltenyi Treg isolation kit.)

I want to sort Treg but I have not yet decided if using the kit or a FACS sorter.

Would you recommend to use a FACS Sorter to get high purity of Tregs?

thanks in advanced

Your yield is low, for sure. Whether you should flow sorting or not depends very mind on what you want to do with the cells. How many do you need and of what purity?

~Hi Cristina, I generally find the purity of the T-reg population is high (>95%) from the MACS kit, purity assessed by FOX-P3 expression, however the 'negative' fraction also contain a fair few Tregs too. I tried flow sorting and whilst the purity was a better for the non-Tregs the yield was similar.

Hi Andrew,

We have been playing with this kit and generally get about 3-5x10^5 pure CD25+ CD127- CD4+ cells per 10^8 PBMCs. Using the LD column is absolutely essential for us as using the LS column results in very low enrichment of CD4+ CD127-/low cells.