Dear RS community,
I would like to determine cytokine profiles of murine splenocytes isolated from infected mice. My plan was to collect spleens from infected and uninfected mice, seed into 24-well plates, stimulate with my (heat-killed) infectious agent and quantify cytokine profiles in the supernatant by Luminex. Theoretically, I would expect a strong response from CD4+ T-lymphocytes, but possibly from DCs or other cells.
Now, I have never done this before so:
1) 24 or 96 well plates, DMEM or RPMI media?
2) Do I need a co-stimulant for T-lymphocytes (ex. concanavalin A)?
3) Do I need a proliferation factor for T-lymphocytes (ex. PHA)?
4) Anything else?
Eventually, I'd also like to sort the cell population after stimulation, and show that, for ex. B and T lymphocytes are proliferating after stimulation with infectious agent.
Thank you in advance!
For splenocyte re-stimulation, I use 96 well plates (1x10^6 cells) and RPMI medium.
I think that if you are re-stimulating in vitro with the antigen you should not need extra stimulation although you should set the conditions, such as antigen concentration, for optimal stimulation. But it could depend on the antigen. I will follow this question for more expert answers...
If you are trying to identify which population produces the studied cytokines, you can try to intracellularly stain for the cytokine, along with surface markers. Note that if you try this you need to treat the cells with GolgiStop to prevent the cytokine secretion.
To assay proliferation you can try a CFSE proliferation method. Maybe this helps: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185625/
Hope this was of help.
Good luck! :)
Thanks! I'm currently testing some variations of the protocol and will update here when I get the results.
Dear Ognjen или боље драги Огњене, as you test some variations of the protocol it’s enough to use 24 well plates in RPMI. Since, I don’t know what the infectious agent you are entered into mice it would be good if in the beginning of experiment do not use the splenocytes from infected mice that'll further stimulate with infectious agent. In this way you can lose the real in vivo effect of infectious agent. First attempt to do intracellular cytokines staining (along with surface markers) after harvesting splenocytes from infected mice (healthy as control)
To be clear, at the beginning of protocol you shouldn’t use costimulant or proliferation factor and see what kind of results you'll get. Later, you make changes in protocol according to the obtained results.
Good luck!
Срећно!
Драги Немања, хвала на савјету. I'm about to run a few conditions in terms of different stimulants and controls as a pilot run. I'll adjust eventually. At the end, I'll have splenocytes from mock-infected and infected mice, age matched, and I'll stimulate them with my heat-killed infectious agent. I'll also use concanavalin A as a non-specific stimulant of T-cells (that should stimulate all T-cell, whether they're matured/differentiated or not. Thanks again.