Recently I'm working on detecting IL22-producing CD4 positive cells in mouse MLN by flow cytometry. However, after using PMA/ionomycin plus GolgiPlug neither 4 hrs or 6 hrs culture, IL-22 positive cells population were still too low to analyse (about 0.5%~3%). Should I need isolate CD4 positive cells by MACS first or are there some tricks to do this experiment?
Thanks!
Hi, are you using naive animals or after some infection? The expression of IL-22 is normally low, the better place to take IL-22+ cells would be the intestine. Nevertheless instead of stimulating with PMA/ionomycin, you can try to use 4 to 6 h of stimulation with IL-23 plus a transport inhibitor, since IL-23 stimulates IL-22 production (some people do this in the lab). Good luck =)
Thanks!
MLN cells were isolated from DSS-treated mice for 7days, and I have tried to use IL23 with PMA/ionomycin/GolgiPlug for 6 h, but, there is still low frequency of IL22+ cells......
Hi,
there is very low frequency of IL-22+ cells, especially in uninfected mice. To get rid of disturbing "trash"-cells, I would recommend to isolate before stimulating with CD90.2 beads or if you just interested in CD4 cells with CD4 positive selection.
Good luck!
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