Hi all, does somebody knows a publication in which is explained at what time point certain cytokines are being produced by splenocytes (BALB/c) when re-stimulated with antigen? We are doing re-stimulation assays with splenocytes and I would like to know after how many hours of incubation I should stop the incubation and measure the cytokine levels in the supernatant. I am interested in cytokines INF-g, IL-4, IL-5, IL-10 and IL-2. Thanks in advance
So, by RE-Stimulation I assume that they have been pre-sensitized to the antigen you are interested in? In that case we have been able to detect cytokine release (IFN-g & IL-2) after 4 hours. After 24 hrs, you should be able to detect the rest of the ones you are interested.
If they have not been pre-sensitized, then we will usually wait 48-72 hours.
Hope this helps :)!!!
Thanks Josh! Yes the splenocytes are pre-sensitized with the antigen. Have you maybe seen an optimal time to stop the incubation after re-stimulation? From experience here we know that for Th2 related cytokines we usually incubate around 72-96 hours, and for Th1 cytokines around 24 hours. But I am trying to find some publications that support that. Thanks again!
I'm not sure the optimal time unfortunately, but you could potentially design a simple experiment to figure it out. Maybe something like this:
Set up several co-cultures in desired conditions in 96 well v bottom plate. At several different time points (maybe 2 hr, 4hr, 6 hr, 12, 24, 36 etc), spin 1 plate down and collect supernatant and store at -20 (I'd say do 1 plate for each time point, rather than collecting supernatant and refreshing media on the same plate). Once you have completed all time points, analyze cytokines from there either by your preferred method.
Thanks, yes I was thinking to set up an experiment like this, I am just hoping that something like this has already been published.
When you look at supernatant after re-stimulation, 24h is definitely the gold standard.
I do not think you need to do any early time point kinetic design. To play safe, you can keep some sample for 48h or 72h.
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