It depends on the question you want to answer. If you want to know what your isolated Th cells do / were doing in the patient, then this is the way to go. If you are out after what the cells might or might not be capable of producing, then you would need to restimulate them with an agent of your choice, being aware that you skew your results by that. But the restimulation gives clearer discrimination of the cytokine profiles the cells are capable of producing.

I would try to go directly and see what is ongoing in the patients without restimulating.

Good luck!

Thanks a lot for your reply Ulf! Yes, I am mostly interested in the contribution of the different Th subsets to the inflammatory state. However, immune cell counts in CSF are relatively low, and I am afraid that cytokine concentrations esp. for IL-17 and IL-10 will be too low to give any meaningful results.

Dear Lina,

I completely agree with Ulf. Ideally, if you have enough cells you could do both with and w/o restimulation in parallel?

I tried to measure ex vivo cytokine production by T cells from the T cell transfer colitis model and while direct ex vivo measurement revealed almost nothing at all, there was considerable cytokine expression (IFNg, IL-17) when I left the cells for 4h with Brefeldin A only to at least allow the cells that have been activated in vivo to accumulate sufficient amounts of cytokine intracellularly in order to be detectable.

Hope that helps.

Hi Lina,

I was wondering how you have managed so far to measure ex vivo intracellular cytokine production from CSF cells without any stimulation. I have done previous CSF flow work in meningitis patients but just looked at surface markers. I am considering some more functional studies but like you was wondering whether I needed to stimulate the cells or not.

Thanks

James

Hi James,

We ended up doing extracellular staining only looking at surface markers just like you. We have minimised the steps in our cell preparation protokol due to low numbers of cells in CSF together with lower ex vivo viability compared to peripheral blood cells.

I would be highly interested in your future experiences, if you attempt staining CSF cells intracellularly!