I am going to be looking for IL-4 and IFNg by ICS and proliferation by Ki-67 expression by flow after ex vivo stimulation with CD3 (5ug/mL-coated plate)+ soluble CD28 (2ug/mL) or PMA (50ng/mL) + iono (1 ug/mL).
I am wondering if I can use mouse splenocytes (then gate on T cells) or if I need to isolate the T cells first (negative selection) for these assays.
Also, for the longer incubation (2-4 days) for proliferation, is it necessary to include IL-2?
If you guys notice a mistake in my protocol, please let me know.
Thanks!!
Spleen cells should work for stimulation. T cell enrichment is not necessary but might work even better, especially if you do CFSE labelling.
It will be difficult to keep cells isolated from mice alive in proliferation experiment beyond 2 days - but you can try IL-2, thats what most people use.
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