I dont understand, how do you know that there is no proliferation? did you do an MTT or Thymidine uptake on the cells. Especially when your media is turning pale that is a sign of active proliferation of T cells.

Do you observe satellite formation?

I use round bottom plates and i do even have to coat it with anti-CD3 and get perfect proliferation, measured by either MTT or thymidine

I measured with CFSE and flow cytometry.  There should be groups of CD4+ cells with progressively lower CFSE staining as they proliferate and when graphed as a histogram, it creates peaks.  There was only one peak in my study and no cells with lower CFSE staining (indicating no proliferation).  

I presume that you guys do CFSE queit often and have the method nailed, but in my own experience flow cytometry depends so much on gating and you gate wrong population or the parameters were set wrong it might give errors.

I still say you should do an MTT method which is easy and dont have to do elaborate method of FACS and this way you can monitor the proliferation for 5 days.

I usually dont change my media and i do 100,000 of T eff and 100,000 of APCs

I would suggest to add anti-CD28 to the plate.

 Most probably you are not properly coating the plates. To check that I would suggest you to stimulate your culture with soluble aCD3. Since you have irradiated APCs in the co-cultures you should not have trouble with soluble aCD3 stimulation. The actual straight of the stimulation with soluble aCD3 in presence of irradiated APC (CD4 depleted splenocytes) will be lower than the stimulation with pb-aCD3, but probably more "physiological".

I never changed the media during the 4 days experiments and never had issues.

Please always have some control wells with the effector cells in number equivalent to the total cell number present in your 1:1 co-cultures Treg/Teff to exclude that the suppression observed is not actually due to media exhaustion (e.i. if you have 50000 Teff + 50000 Treg you should have a control well with 100000 Teff cells).

Good luck

For a proliferation assay, stimulating with CD3/CD28  beads will assure T cells proliferation, and there is no need to refresh the media, I use to culture my cells for 4-5 days with no problem and observe a nice proliferation with CFSE.

Good luck

Hi Miranda,

If you have used CFSE at a concentration of (0.5ug/ml) for in vitro assays, the cells will survive. If you go beyond this concentration, CFSE (even 1.5 ug/ml) is very toxic to the cells and that by itself will kill all your Teff cells and hence you cannot expect any proliferation. So I wonder at what concentration you did your CFSE labeling. If you repeat the assay, please stain CFSE at 0.5ug/ml and not at any higher concentration. Your assay set up looks good. So I doubt only the CFSE labeling concentration. Also you could check the Treg suppression inspector page (Miltenyi Biotech) which gives a detailed description of assay setup. Wish you all the best!

Hi all.  Thanks for your amazing advice and input.  I tried another experiment with just the Teff cells to see if I could just get them to proliferate and still couldn't get them to.  I tried APCs + plate-bound CD3 +/- soluble CD28.  Our CFSE staining concentration is 0.5 ug/ml.  I'm going to try ordering new CD3 and CD28 antibodies this time and try it again to see if those are possibly the problem.  I'll keep you all posted!

Just as a note,  if you use anti-CD3e / CD28 coated beads as suggested by Kawthar,  you should get proliferation but Treg suppression will not occur.  I have had good success with Treg suppression assays using B6 T cells but Treg suppression only works when there are bona fide APCs in the co-culture,  not just beads.   I published a protocol for the assay you're trying to do earlier this year actually,  just with CTV instead of CFSE. See http://www.bio-protocol.org/e1698

Concerning T cell proliferation assays with plate-bound CD3/CD28 antibodies: make sure that you use approriate cell culture plates: some do bind the agonistic antibodies, others not. I found that e.g. Corning Costar 3997 plates do work but Falcon 353072 plates do not work for such proliferation assays. In case of Treg suppression assays however you should not have trouble with plate binding properties if you use irradiated splenocytes as APCs: in this case soluble anti-CD3 stimulus alone should be sufficient to induce proliferation.