Dear all, To observe the immunosuppressive function of Treg, I will perform two different approaches of T cell proliferation assay. 1. 1.10^5 Splenocytes from mice labeling with cell trace violet (CTV, to observe their proliferation) stimulating with 5 ug/ml Concanavalin A in a P96 flat-bottom (100 uL/well of RPMI) for 3 days of culture. I already run some tests and I was able to see beautiful peaks of proliferation but a high number of cell deaths. 2. 1.10^5 Splenocytes from mice labeling with cell trace violet (CTV, to observe their proliferation) stimulating with CD3/CD28 dynabeads (ratio 1cell:2 beads) + IL-2 (100 units) in a P96 flat-bottom (100 uL/well of RPMI) for 7 days of culture. Them after 6 hours of stimulation I will add the Tregs. I'm wondering if it is better to purify T cells for these tests using the negative selection and magnetic LS column before starting the proliferation assay. For that I will use the biotin that I already have: CD11b, CD19 and Ter119, from previous experiments so I don't need to buy more. My question is if this protocol is correct and if a cell isolation step should help get rid of the cell populations that will likely die away because they are not stimulated and if this purification could impact on the Treg mechanism of immunosuppression. Thank you and have a great day,
Hi! Ideally the Treg cell isolation step should not cause a change in the property of causing immunosuppression. However, with cell isolation protocols the main caveat that a lot of people face is a loss of cell numbers. Since the Treg numbers are low endogenously, carrying out an isolation protocol could lead to further reduction which could overall change the phenotype of the assay. I would only suggest that you could compare the Treg numbers before and after isolation to understand if the numbers are approximately the same before performing the proliferation assay. Thanks!
Thank you Sanmoy Pathak for your reply.
I'm looking for the answers for the proliferation assay and if in the culture it is important to have B-cells, NK cells, etc to Treg's mechanism of immunosuppression.
Best,
Hi Aline D. de Lima ,
I would recommend purifying the T cells mainly so you will have enough events to analyze. There is cell death in these assays always but you should just gate away from it using a viability dye. We always used a viability dye and CD8 staining so that we would not be picking up the CD4+ T reg. I don't know what you are using as a source of T reg. You should use different ratios of T reg : splenocytes/T cells to get an sense of the potency of the T reg.
There's a protocol at the website of my former company, Cellero. Check it out before they take the site down.
Dear Aline D. de Lima ,
Its always better to isolate/sort T cells in such assays (if your resources allows; In my place resources does not allow so we get going with whole cells), so as to remove any kind of confounding effect of rest of the populations. Although these cells does not effect much in CD3/CD28 or ConA stimulation condition.
Dont bother about the cell death as in such assays, as the mitogen cause death, which may easily be removed by live cell gating (which you are doing).
Jitendra Kumar Shandilya Thank you so much for your ansewer,
Best regards,