I am trying to keep cytokines expression inside cells by adding berfeldin A in the cell culture. Unfortunately, berfeldin A is not stopping cytokines secrection, and I have checked that by ELISA.
How do you know brefeldin A is not stopping?
You need to have a control, that is without brefeldin A and compare the ELISA for both (with and without brefeldin A). If you observe no difference, then you assume brefeldin is not working.
I assume brefeldin A can not stop the secretion 100%. And it depends also on the duration of stimulation and incubation of the cells with brefeldin A.
I hope this helps. more information is needed to help you better. Goodluck
Brefeldin A and monensin probably do not completely stop excretion of your cytokines. In addition to timing of exposure, concentration is also something you could titrate (as we observed below a higher concentration of monensin correlatd with a higher proportion of T cells positive for IFN-gamma but not IL-10).
Article Intracellular IL-10 detection in T cells by flowcytometry: T...
Detection of intracellular cytokines by flow cytometry in presence of protein transport inhibitors depends upon the kinetics of the cytokines under investigation. As discussed earlier the monensin or Brefeldin may not be able to stop the cytokine secretion completely. For IFN gamma, TNF alfa, and IL17 stimulation for 4-6 hrs in presence of Brefeldin A is sufficient. The concentration I used is 5 micrograms per ml of medium in 4 well plate for 1 million cells. Brefeldin A should be reconstituted in DMSO.
Hope this will help
Thanks
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