I'm currently trying to stain for intracellular cytokines in mast cells, and have been having trouble getting my stains to show up by flow. I know that these cytokines are being produced, as measured by ELISA and RNA. I've also done nearly this exact same assay in B-cells without any issues, with a very clean dose-response curve. However, in B cells, I used a different stimulation, and I'm not seeing any of the cytokines I'm measuring by flow at all in a bone-marrow-derived mast cell population. My protocol is as follows:
1: Wash mast cells, plate an appropriate amount
2: Add Golgistop as per manufacturer's protocol
3: Add stimulation immediately after, incubate 4 hours at 37C
4: Stain, perm, stain, etc.
5: Run flow
Given that TLR4/LPS often needs to be internalized, is it possible that by adding Golgistop before the stimulation, I'm blocking that signaling? I'm thinking I will try to add golgistop after 2 of the 4 hours of my stim, but does anyone else have any ideas as to why I'm not seeing IC cytokine staining?
Edit: The only marker not showing up after staining is my IC cytokine marker. Everything else in the assay works fine.
Hi Ethan,
I would stronlgy suggest to add Golgistop SUBSEQUENT to LPS (as you mentioned)... However, you should consider also:
i) time course for different cytokines produced is quite different (ranging from 1h to few hours). Thus, I would suggest to perform a pre-test WITHOUT Monensin and testing the appearance of your cytokine of interest in the supernatant. Thereby you can define the optimal time point (shortly before Maximum in the supernatant) for ICCS (LPS total ...hours [for the last 2h in the presence of Monensin])
ii) Monensin might not be the appropriate Golgi-Block: detection of some cytokines (like Il6) by ICCS is much more reliable with Brefeldin A. Thus, you might do the time course (i) and than stain your cytokines of interest in cultures that were treated with Monensin vs Brefeldin A side-by-side
Good luck!
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