I want to incubate human CD4 T memory cells for 48 to dterminate cycle entry and proliferation. I plan to stimulate them for 48 hours. Can I add brefeldin for the last hors of this incubation in order to detect intracellular cytokines by flow cytometry?
This is doable for cytokines but not if you are looking at T cell proliferation. T cells need at least 3-5 days to start proliferating and the best time point would be 7-8 days post stimulation. However, if you are looking at intracellular cytokine production, you could go for up to 72 hours in my experience (probably longer). You just add BFA/monensin for the last 4-5 hours and then do flow cytometry. Signals tend to be better at early time points though, esp. for TNF. If you need to correlate T cell proliferation with cytokine production, do ELISA/multiplex on the cell supernatants at D7 when you harvest the T cells.
Hi Enrique,
I think Julie cover pretty good insight into this type of assays. The only thing were I might disagree is the timepoint for looking at proliferation but this largely depends on your type of stimulation.
Is you stim anti-CD3 and anti-CD28 +IL-2? If so, I've done this with mouse T cells and you can see substancial division 3 days. I'm pretty sure the kinetics of hu T cells would be pretty similar. If you could pre-enrich for your target population prior to stimulation would be good as this kind of treatment heavily downregulated CD3/TCR, CD4 and CD8 wich makes it a bit tricky to gate on later.
With hu memory CD8 T cells stimulated with peptide we used to expand PBMCs for 10 days before a second peptide/restim to look at cytokine production, there can be a little bit by d7 but probably not worth to look at yet. You could check proliferation of these cells too, probably not before day 7, but you'd need a tetramer to detect the antigen specific cells.
Good luck!
Thank you very much, Sergio and Julie.
You gave me a starting point.
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