I just try to analysis the Th17/Treg imbalance from isolated LPMC use flow cytometry. For analysis of Th17, cell activation ( stimulated with PMA and ionomycin) is often required. But for analysis of Treg, is it necessary to do the procedure of cell activation? Can I skip the step for further staining?
Hello,
You don't need to stimulate Tregs with PMA and ionomycin in order to identify them - intracellular staining for Foxp3 protein expression is sufficient.
The purpose of PMA and ionomycin in your setting is to activate the T cells in your sample so that they synthesize cytokines (such as IL-17) that can then be identified by intracellular staining. Foxp3, a transcription factor, will be present in Tregs whether or not they are stimulated with PMA and ionomycin and so this stimulation is unnecessary.
Having said this, the design of your experiment and/or how much sample you have will dictate whether you can actually stimulate, stain and analyze the cells separately for their content of Tregs and Th17 cells.
Hello,
You don't need to stimulate Tregs with PMA and ionomycin in order to identify them - intracellular staining for Foxp3 protein expression is sufficient.
The purpose of PMA and ionomycin in your setting is to activate the T cells in your sample so that they synthesize cytokines (such as IL-17) that can then be identified by intracellular staining. Foxp3, a transcription factor, will be present in Tregs whether or not they are stimulated with PMA and ionomycin and so this stimulation is unnecessary.
Having said this, the design of your experiment and/or how much sample you have will dictate whether you can actually stimulate, stain and analyze the cells separately for their content of Tregs and Th17 cells.
Like is said before: direct FoxP3 staining (without stimulation) seperated from your IL-17 staining after stimulation... depending on your sample size (if you do have enough cells); but if you are interesting in different IL-17 producing cell subsets... maybe it is also interesting to combine the IL-17 stain together with the FoxP3 stain.. could be a hint for some diseases?!
Referring to:
Pathogenic conversion of Foxp3+ T cells into TH17 cells in autoimmune arthritis
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