Hi,
I'm having trouble trying to stain mouse IL-4 from Th2 cells polarized in vitro.
I've been purifying CD4+ cells from BABL/c spleens with magnetic beads and culturing them in CD3/CD28 pre-coated wells in presence of IL-2, IL-4, aIFNg and aIL-12. At day 2 I add fresh media complemented with IL-2 and leave them for 3 more days. Finally, I harvest the cells and stain intracellular cytokines for ulterior FACS analysis.
In polarized Th1 cells I ussually get 40-70% IFNg producing cells, but I get no mark at all for IL-4 in Th2.
PS: Th2 seems to differ morphologically and present differential lectin binding from undifferentiated controls (Th0, cultured in media only with IL-2). I'll check it anyway out by ELISA (discarding supernatant before media change to make sure the IL-4 is secreted from the cells), but I really need to detect IL4 producing cells via FACS.
Any suggestions? Someone had the same issue?
Thanks for reading,
Hi Martin,
Whats your Th2 differentiation condition? Whats your staining protocol for intracellular IL-4 ( are you using Brefeldin A/ Monensin to block cytokine secretion?). Have you considered looking at earlier time-points for IL-4 production? To confirm that its not an issue with the IL-4 staining protocol, i would reccomend trying qpcr as a cheap alternative to validate its not a staining issue.
Thanks Simon for your answer,
Th2 differentation condition is: IL-2 (20 ng/ml), IL-4 (50ng/ml), aIFNg (10ug/ml) and aIL-12 (10ug/ml).
At the last day, I re-stimulate cells with PMA/Ionomycin alone for 2 hours and then add Monensin and Brefeldin A for 4 more hours.
Wich time points do you suggest?
It seems that you are doing all the right steps. The conditions of differentiation, PMA, Brefeldin e.t.c all seem to be in line with what we do. My recommendation would be to check the IL-4 antibody (if there is a lab next door that can lend you a different one that would be ideal). It might not be a bad idea to do qPCR as well looking for IL-4 levels (this would also address the issue of the IL-4 antibody not working).
Hi,
i think I would try several things:
1. Coated plates can be tricky. I prefer coating them with CD3 and add soluble anti CD28. Also, you can try using CD3/CD28 beads instead.
2. When you're adding fresh medium, you're diluting the aIFNg and aIL12. I think you should add them again to the same final concentration.
3. It wasn't clear to me if you re-stim them before you're doing the IL4 ICC staining. If not then you should try 5-6h stim with PMA and ionomycin in the presence of a golgi plug and then do the staining. Since there should be some Th2 cells in the spleen, I would try first simply stimulating fresh splenocyte with PMA / iono for 5h(+obviously with golgi plug in the medium), just to see if your staining protocol works before you're wasting more time and reagents.
Good luck,
david
Hi Martin, have you also checked for IL5 and/or Il13 in your Th2-polarized cells? Stainig for those 2 cytokines always works a bit better than staining for IL4 in my hands. Besides, I would agree with Simon that IL4 antibody could be the most likely problem, rather than the protocol as it seems OK.
On the other hand, I have to disagree with David on his 3 point: the precentage of IL-4 producing Th cells in spleens of healthy mice is very low and I would not recommed using those for checking your protocol.
Hello everyone,
First of all, thank you for your valuable answers.
David, I re-stimulate cells with PMA/Ionomycin alone for 2 hours and then add Monensin and Brefeldin A for 4 more hours (that's 6 yours total for PMA/Ionomycin)
I'm concluding the antibody (Biolegend aIL4-PE [Clone 11B11]) is somehow failing... have you ever tried this one before? I´ll check out for another clone or cytokine I guess.
Antibody: (http://www.biolegend.com/pe-anti-mouse-il-4-antibody-893.html)
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