Hello all,
I am trying to stain for IL-17A in normal human PBMCs. I have used 3 different methods to stimulate my cells. 1) PHA (24 hours), 2) PMA/Ionomycin/Brefeldin A (5 hours), and 3) cell stimulating cocktail from Tonbo Bio (5 hours) to stimulate the cells, but I could not detect IL-17A by flow cytometry with all the methods. Does anyone know what might be happening? Any help or suggestions are welcome!
Thank you!
Lin
Hi
Find PDF attached ( ,Frequently Aske question in Detection of Cytokines )
Published by BD Biosciences. I hope helping you.
Good luck
It depends on what type of cells you are focusing in PBMCs and the sensitivity of reagents used. You have used the correct positive controls. We should recognize that all regulatory pathways need not employ cytokines! The following back-to-back papers in recent J Immunology 197:(3), 08/01/2016 from Shane Crotty's lab used a new Activation-Induced Marker technique to look for cytokine-independent pathways. They looked for IL-17 as well although not in PBMCs. Thought, it would be worth checking.
Hi,
I recommend to stimulate with Staphylococcus enterotoxin B (SEB), e.g. Sigma S4881. I use it at 100-200 ng per mL of whole blood or PBMC.SEB is a so called superantigen and stimulates approximately 20-30% of the human T lymphocytes by covalently binding TCR on T lymphocytes and MHC on antigen presenting cells. Using SEB I regularly see small but definite T cell subpopulations with intracellular IL-17, IL-10, IFNγ etc.
Good luck,
Axel
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