Hello Ashley, your question isn't very clear concerning your protocol. You are using irradiated total NOD splenocytes as Ag presenting cells and TCR Tg on NOD background splenocytes containing your Tg CD4 T cell population + all other cells non irradiated?

did I understand right?

Hi Ashlee: I agree it is not clear which is the antigen your T cells must recognize. But anyway I will try with spleen dendritic cells that you can sort or isolate with MACS-CD11c, this cells will be good antigen presenting cells. Good luck.

U should compare BM DCs to splenic DCs to see where you can get the most potent T cell activators.  If by antigen specific you mean you will load DCs with prostate derived antigens well then you probably need to optimize your antigen loading protocol.  Additionally, a problem may be that these T cells have been anergized and will not respond.  Have you tried assessing their activation potential, PMA/Ionomycin or anti CD3 beads?).  You may have non-functional T cells.

Sorry, that I was not clear about the protocol I am using, George and Maria. The protocol, which I am using, in the paper I cited is listed under the heading "Proliferation assay". I am not using any transgenic mice. So I am using irradiated total NOD splenocytes as Ag presenting cells, which have been incubated with NOD mouse prostate lysate + (hopefully) antigen-specific splenocytes from a NOD mouse.

Thank you, Oscar! I am currently trying to see if anti-CD3 and -CD28 Ab can cause expansion of my splenic T-cells. Also how would I go about optimizing an antigen loading protocol? As in how can I check to see if my splenocytes are actually loaded with prostate antigen without incubating them with T-cells and then checking proliferation of the T-cells?

Hi Ashlee, going by your last statement, i believe you are trying to "expand a specific population of T cells".

I am attaching a publication of mine with the protocol to expand single cell T cell clones, done in NOD mice. The trick is to be patient. Generally, you will get 4-5 wells/96 wells with some cell growth and survival. So start with 4-5 96W plates, plated at 1 cell per well. Also use U bottom plates. Once you have cells growing out, transfer them to 2/3 96W plates, then 48 w plate, 24 w plate, before you can finally expand them in 6 well plates. I would suggest making freeze downs in the 48 or 24 well plate stage. Finally, do not over stimulate the cells. Use half the peptide concentration that you would use for bulk stimulation while cloning.

Good luck

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