I'm looking for a positive control for IL-4 secretion in a 5 - 7 day PBMC proliferation. Currently I'm stimulating the cells with CD3 (0.2 ug) in the presence of IL-2 (10 ng) for the culture with further stimulation with PMA/ionomycin/BrefA on the final day (around 6) hours and then attempting to detect IL-4 in Th2 cells. So far very little IL-4 signal (lots of IFNg), and I'm not sure if my stimuli or staining protocol is the main problem. Any thoughts on either would be welcome!
Hi Amanda-
You're not alone on the IL-4 front! It seems to be a tricky cytokine to monitor, and I had similar struggles. I published an OMIP in Cytometry that contains data on stimulated PBMC and production of IL-4. This protocol was based on suggestions from BD, eBioscience, and primary literature. Take a look at Online Figure 2, which gives the details. I believe there are other OMIPs that address IL-4, and I'm sure there are other protocols out there too. I'm not saying my protocol is the best, but it worked in my hands. Let me know if you have any questions, or if there's any other help I can offer.
http://www.ncbi.nlm.nih.gov/pubmed/?myncbishare=uwisclib&otool=uwisclib&cmd=Search&term=omip-008&submit2=Go
According to Katial et al, which evaluated different mitogens on immunocompetent donor PBMC, pokeweed mitogen (PMW) induces the most increased production of IL-4. Though the reference is old (1998) you might as well check it. Here is the link:
http://cvi.asm.org/content/5/1/78.full
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