I have been trying to look for intra-cellular TNFa and IL-12 in granulocytes. For this, I have used Golgi-Plug (Brefeldin A) to block cytokine release. Unfortunately, it looks like either the cells don't respond to my stimulation or Brefeldin A does not block cytokine release from granulocytes (perhaps because they use different secretion/release mechanisms).
If anyone has any experience with intra-cellular cytokine staining of neutrophils, I would really appreciate any tip and trick! Thanks!
Hi Damien. Are you using a permeabilization/fixation step before the staining? What are you using as control to see if Brefeldin A is working correctly? This information is needed to give you a more precise approach. In addition this might help: http://www.jleukbio.org/content/68/1/125.long
Good luck
Dear Ivan, thank you for your answer. For fixation, I use a kit from BD with a CytoFix-CytoPerm solution. In this experiment, I am actually staining a mixed population of bone marrow cells after a short stimulation. I know Brefeldin A works in general, because other cell populations show good intra-cellular stainings. Only granulocytes seem to either not respond to the stimuli or cannot be either blocked for cytokine release or stained intra-cellularly. I was thinking of trying to use GolgiStop (Monensin) instead of GolgiPlug (Brefeldin A). I am just not sure that one can really block the release of cytokines from neutrophils. Any tip or tricks would be greatly appreciated! Thanks.
Hello Damien, I am not sure that BFA or Monensin will work on granulocytes because many cytokines are stored in the secretory granules present in the cytoplasm of neutrophils and eosinophils prior to release by regulated exocytosis. They are very different from lymphocytes which synthesize cytokines de novo through a constitutive secretory pathway, which is sensitive to Golgi-blocking proteins (see attached link). Both of those drugs block transportation of proteins from the Golgi and will not have an effect on granule-stored cytokines, as granules contain already synthesized proteins that are waiting for receptor-triggered release of their contents. Hope this helps, Paige
http://www.bloodjournal.org/content/118/1/9.long?sso-checked=true
Dear Paige,
Thank you so much for your answer. This is exactly what I was afraid of. I still asked around in case somebody knew a trick. I find very hard to characterize the response of granulocytes. They seem to really not like to be purified before stimulation (via MACS sorting or Percoll gradient). This is why I have tried to look at intracellular cytokines in the mixed population. Unfortunately, nothing is simple ... I will have to try to find another way.
Anyway, thanks again for your input and for the review. Really interesting!
Damien
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