I'm looking for T cell proliferation assay setup.
Has it a rationale to use splenocytes from two congeneic mice 1) as responders (from immunized mice, cfse labeled) and 2) as APC from the second in T cell proliferation assay? (gating on congeneics and CD4)
As far as I searched, I couldn't find this combination, purified T cells (responders)+splenocytes - yes, just splenocytes - yes, with the engagement of cell lines - yes, but with two splenocytes from two source in antigen specific proliferation assay - at present - no.
Why if gating is used, one doesn't use whole splenocytes (erythrocyte lysed of course)? The only hint I have found is "high background". Is it indeed obligatory to separate T cell responders?
Why I'm not concerning spleen from one animal (immunized), because (if the logic is correct) I will know the exact time when I added responders (antigen needs processing, so one need to wait) when I use two spleens.
Second question inside: Odoes mitomycin affect antigen processing, is it obligatory to add it after presumed antigen full processing?
Hi Vera:
From a purely FACS perspective you can clearly gate T cells from a specific source if you mix whole splenocytes with different cogenic background (e.g. CD90.1 vs CD90.2 [aka Thy1.1 vs Thy1.2] or CD45.1 vs CD45.2).
The reason to not do that is not technical noise/background, but analytical.
From a immunologic perspective it is not proper. Here is the thing: I assume that you're willing to evaluate specific features of the T cell compartment (a typical example is to use TCR Tg but also cogenic CD90.1 T cells). Or alternatively, which does not seems to be your case, specific APC properties (pre-stimulation features, Tg expression of signals 2, amongst other).
So, for example, if you just mix CD90.1 CFSE labeled Tg whole splenocytes with whole unlabeled CD90.2 splenocytes supposed to play the role of APCs, then you can certainly do an accurate CFSE dilution analysis, but you cannot know the influence of the activated UNLABELED CD90.2 T cells, which might, for instance, engage the syngeneic unlabeled CD90.2 APCs and be an important source of IL2, thus influencing the CD90.1 outcome. The scenario become even more complex considering that the CD90.1 APCs might crosstalk with both cogenic T cells, and viceversa. Either if you will to evaluate specific T cell response or APC capacity, you won't be able to discriminate the influence of each population of whole splenocyes.
So, if you're willing to dissect influence of specific cellular subsets, then you must enrich/purify each sample for the desire type of cell; not for technical/FACS reasons, but for analytic reasons.
To mix whole splenocytes from different cogenic sources will not allow you to make better immunologic analysis than just culturing splenocytes from a single source, despite in the former case you can gate specific cogenic subsets.
Hope my explanation was clear enough. Otherwise feel free to reply.
Kind regards
William
@William Bracamonte
Thank you! Now I realise the reason.
But the mice are not TCR Tg.
"...but you cannot know the influence of the activated UNLABELED CD90.2 T cells, which might, for instance, engage the syngeneic unlabeled CD90.2 APCs"
but in principle it can occur if I use unseparated spleen from nonimmunized mice as a source of APC.
But yes, mix of two spleens is not a good choise.
Dear Vera, agree to William. I would recommend the simplest way, culturing CFSE-labeled splenocytes from immune mice with different concentrations of antigen during 3-4 days. Then, you can use staining with monoclonal antibodies to subpopulations of interest to evaluate their impact in proliferation. If you have antigen in recombinant form, you should take into account possible presence of microbial LPS in antigen preparations, which can induce nonspecific proliferation of B-cells. This non-specific proliferation may be main source of "high background" levels. Obviously, you can avoid this by reducing concentrations of antigen. The best way would be to use preparations of antigen specifically purified from LPS.
@Dmitry B. Kazansky Thank you for the response.
Hope eventually will end up with purified T cells (immune) and splenocytes.
One more question: if mitomycin c treatment can substitute gamma-irradiation, and antigen needs to be processed (not in peptide form already), mitomycin treatment should be performed after presumed processing (e.g. after 24 h)?
I think, yes. Stimulators should not proliferate. MitC does not affect processing. Note, that treatment with MitC will require subsequent intensive washing of cells, no less than 3 times. Third centrifugation of stimulators would be better to perform after 20-30 min incubation at 37 degrees in washing medium.
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