I am going to evaluate the preventive effect of probiotic in C.rodentium induced colitis. Especially I want to test its ability in restoring the imbalance of Th17/Treg. First, I will use flow cytometry to obtain the Th17/Treg ratio from isolated LPMC. But I am confused about how to choose the exact method to detect the Th17/Treg related cytokine. Do I use ELISA , RT-qPCR or both?
If I use ELISA how do I choose the sample from peripheral Blood, cell supernatant or tissue homogenate?
Is it necessary to analysis the Th1 and Th2 related cytokine? I 'm a rookie in IBD research so I hope to obtain your experienced guidance.
If you want to detect the cytokines produced by Th17/Treg you should do an intracellular staining for your expected cytokines (IL17a IL22 and all the other RORgammaT related cytokines). You stimulate your lymphocytes with specific cytokines or with PMA-ionomicin and after 4/8/18/24h of stimulation you stain your cells adding some antibodies for your candidate cytokines in the panel.
ELISA or RTqPCR can be valid tool as a preliminary study to do on the tissue and to determine which cytokines can be your candidates, as you can have an idea of what is generally produced. Although I would rather use some publications as starting point, there are so many articles describing Th17/Treg and ILC3 cytokines produced in the gut.
If you want to detect the cytokines produced by Th17/Treg you should do an intracellular staining for your expected cytokines (IL17a IL22 and all the other RORgammaT related cytokines). You stimulate your lymphocytes with specific cytokines or with PMA-ionomicin and after 4/8/18/24h of stimulation you stain your cells adding some antibodies for your candidate cytokines in the panel.
ELISA or RTqPCR can be valid tool as a preliminary study to do on the tissue and to determine which cytokines can be your candidates, as you can have an idea of what is generally produced. Although I would rather use some publications as starting point, there are so many articles describing Th17/Treg and ILC3 cytokines produced in the gut.
Hi Chen Xin Jun
I prefer to evaluate the cells ex-vivo rather than stimulated with PMA/Ionomycin, but these are an option if the frequency of these cells are low. If you are going to stimulate the cells you could measure the cytokines of Th17 (IL-17A, IL-17F, IL-21, IL-22) and Tregs ( IL-10 and TGF-b) in the cell supernatant. You can measure the Th17/tregs cytokines in the serum from peripheral blood, but I think that the site of inflammation is on the intestine, therefore, is better to measure the mRNA of these cytokines in situ from the tissue. I think that your work are complete with the analisis of Th17/Tregs and you do not need to analize the Th1/Th2 cells .
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