You incubate for minimum of 2h before Brefaldin A to allow for sufficient antigen presentation to occur.
Hi Edward,
Brefildin A will affect intracellular vesicule trafficking in all cells in your well. If the peptide presentation is independent of the arrival/recycling of MHC molecules on the surface of the antigen presenting cells in your model, the addition of Brefildin A will not affect the activation of the T cells.
I wouldn't recommend it though. I'd give more time to the APCs to capture and load the peptide correctly before adding the Brefildin A.
If your antigen is a full protein Brefildin A will definitely affect the processing and the presentation so I wouldn't add it before at least 6h of co-culture.
Hope this helps,
Good luck,
G.
Brefeldin will prevent cytokines are secreted. So it depends on the question you want to answer. If you do in vitro stimulation with peptide will need to put brefeldin.
HI Edward, I agree with answer of Georges Azar. It is better to give more time for stimulation before adding Brefeldin A (BFA). And BFA should not be kept in cell culture for longer than 12h. In my experiment, BFA was added during the last 6h.
Hope this answers your question. Best
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