Dear, In my experimental model I will administer a substance intraperitoneally and some time later I need to collect the T lymphocytes from the spleen for phenotypic characterization in order to evaluate the host's adaptive immune response. I need to know: i) on average, how long after administering the substance I should collect the spleen; ii) how to isolate T cells from spleen and, finally, which T lymphocyte activation surface markers I should use (in addition to CD8; CD4 and CD69).
Hey!
I am not sure about how long you would need to wait after the injection but in general you can use CD3 dyna beads to isolate T cells. Then stain for CD4 and CD8 to analyze separate populations. For activation you can use CD25 or CD69 antibody.
Hope this helps!!
If you only want to look at activation of T cells directly by FACS (i.e. no plans to do T cell cultures) I would not do a purification of T cells (expensive, time consuming, potential loss of cells) and stain for CD3, (gd and ab T cells, NK T cells) and TCRab (if you want to focus on ab T cells only) and CD69 and CD25 markers. CD69 comes up after around 4 hours (and diminishes over time), CD25 after 16 to 24 hours. Other markers could be PD1, TIM3, cytokine receptors, etc. It may be interesting to also look at T cells in the peritoneum (attracted by or activated by your substance). You can isolate them by euthanizing the animal and injection 10 ml of PBS i.p. with a fine gauge needle, massage the belly and recuperate the liquid wit a wider gauge needle.
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