with anti CD3/25 you will test for the overall T cell potential to respond. This is not what you want. Therefore, re-stimulate with the immunogen and use elispot or IFN-g in the supernatant of 48h cultures as read-out.

If you want to use anti-CD3/CD28 for a round of activation before going on Flow, as a common practice in science you will have your negative control of the same cells activated with anti-CD3/CD28 without any antigens. That should show if there is a shift in activation/re-activation in this case. 

Another way you could use if your antigen-specific T cell response are rare is by using ELISPOT assay. That way you can detect as low as 1 cell from 1 million cells responding towards your antigen. And I don't re-activate them with anti-CD3/CD28.

Hope that helps!

Thank you for your answer Norkhairin Yusuf, it is very helpful.

 I would avoid adding any anti-CD3/CD28 to my samples immunized in vivo. Anti CD3/28 would activate every T cells without the need for specific antigen, so it would defy the purpose of your experiment. If you know your peptide or even protein antigen go ahead and use ELISPOT assay. I have attached a paper from my lab using this technique. ncomms6369-s1 includes supplementary data for the ncomms6369, in case you need it. Curr.Op.Imm paper is a review but does have another ELISPOT experiment. Goof luck.

with anti CD3/25 you will test for the overall T cell potential to respond. This is not what you want. Therefore, re-stimulate with the immunogen and use elispot or IFN-g in the supernatant of 48h cultures as read-out.

I will re stimulate my cells with the antigen (short overlapping peptides if available) and add CD44 antibody to the flow panel.  The CD44 will allow you to gate on antigen experienced T cells,  which will help capture the actual percentage of the vaccine antigen T cells producing IFN-g, IL-2, TN or whatever cytokine u like.ELISPOT is highly sensitive but flow has many advantages too.

Flow will also help you determine the type of T cell responding to your vaccine. Something you can't do with ELISPOT unless u presort your cells.

If you prefer flow cytometry (instead of the suggested ELISPOT) you migth detect actual ANTIGEN-Specifc T cells (and expressed cytokines simultaneously) by tetramer staining (if you are interested in CD8+ CTLs) or by CD154 staining (if you are interested in CD4+ Th cells) - in the latter case refer to

PMID:

16186818, 16186817, 17406204, 18785645

Thank so much everyone, your input is certainly very helpful!!.

I would avoid using anti-CD3, as you only want to detect antigen-specific T cell responses. All you need to add is the antigen. However, sometimes the addition of anti-CD28 can improve your IFN-g production (especially if your incubation is short-term), without increasing the background non-specific responses. Just make sure you have the right controls (unstimulated T cells , T cells stimulated just with anti-CD28 and T cells stimulated with antigen+anti-CD28)

Good luck