I'm staining mouse splenocytes for Foxp3 using the eBioscience kit. I'm also staining the same cells (different tubes) for IL-17 and IFNg so I do a PMA/ionomycin/Brefeldin A stimulation on all the cells first. I upped the PMA and ionomycin for this new model we are doing and now I get squat for Foxp3. This could also be an old antibody or just a lack of Foxp3, but I'm curious what others have found.
Thanks!
Standard concentrations are PMA (50 ng/mL) and Ionomycin (1 ug/mL). Addition of anti-CD3 increase percentage of FoxP3+ cells. How long do you activate the cells?
I would simply suggest magnetic isolation of CD4+ splenocytes and test/titrate your FoxP3 antibody.
This should not affect the detection of FOXp3. Best would be to titrate your antibody.
I have never ever experienced the same. which was the fluorochrome used for Foxp3? what was the procedure? stimulation-surface staining-fixation/permeabilization-intracellular staining?
Standard concentrations are PMA (50 ng/mL) and Ionomycin (1 ug/mL). Addition of anti-CD3 increase percentage of FoxP3+ cells. How long do you activate the cells?
I would simply suggest magnetic isolation of CD4+ splenocytes and test/titrate your FoxP3 antibody.
Hi Emily,
We are able to perform intracellular cytokine staining with Foxp3 staining for mouse cells without issues for IFN-g & IL-17A. We add the anti-IFN-g & IL-17 in the same permeabilization buffer with the Foxp3 antibody for staining and pipet mix to ensure good resolution on the stain. You may wish to check how old your perm buffer is. At iQ Biosciences, we do not exceed the expiration date and make those solutions fresh per experiment. Although it is tedious, it helps avoid those annoying issues when troubleshooting.
Good Luck,
Omar
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