I'm hoping for a little help with a Treg proliferation assay using cell trace proliferation dyes.I started by sorting responder T-cells (CD4+CD25-) and Tregs (CD4+CD25+CD127low; then labeled freshly isolated Tresp with CFSE. I added Tresp and Tresp cells at a 1:1 , 2:1, 4:1, 8:1and 16:1 ratio in a U-bottom plate (using 4,000 cells) and then add CD3 CD28 to simulate. I had Tresp wells without Treg as positive controls and without anti-CD3/CD28 as negative controls. Cocultured for 4 days. Looking at the control wells, I had got no proliferation of the Tresp without stimulations and lovely proliferation peaks with stimulations. However, my problem is that the suppression was increase with the reduced ratio. that means at the ratio 1:16 the suppression is highest, while at the 1:1, the suppression is lowest. I am wondering it is normal in human beings?
Any help/advise would be much appreciated.Thanks
Hi Liu. Did you check FoxP3 expression in your CD4+CD25- and CD4+CD25+ cell populations? If there is contamination of Tregs in effector cell poplation or that of effectors in Tregs, increasing ratios may affect the outcome of the assay.
Dr Sharma,
Thank you so much for your reply. I didn't check the Foxp3 expression. I will try later. I encountered another problem. The positive control (add CD3 and CD28, without Treg)had no proliferation. And the cocultrue wells including treg and effectors cell also had no proliferation. But unstain well and CFSE FMO wells (NO CFSE staining, no treg , add cd3cd28) had obvious proliferation. so I wonder the problem may due to CFSE labelinng was to high. Then I reduced the cons. of CFSE to 2 um, this time except the positve and negative control wells, others wells have proliferation. The problem is why the positive well had no proliferation, and also same problems suppression was increase with the reduced ratio ?
Could you help me to check where is the problem again? Thank you so much for your kind help in advance
Hi Liu. Will you be able to share the flow cytograms and your CFSE staining protocol? We have always used 5uM CFSE and it works well.
Unstained well and CFSE FMO well isn't the same well?
How did you check proliferation in wells without CFSE? By flow cytometry (FSC Vs SSC) or by microscopy?
I will suggest to double check your CFSE concentration also. Will be better able to understand after looking at the above queries.
Good luck
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