I am trying to stimulate the PBMC from a healthy donor but its not happening. Do the healthy PBMCs get stimulated? I used PMA/Iono/LPS and IL23 overnight. Are there any tried and tested protocols that someone could share with me?
Hi Sharat
The PBMC from healthy donor can be stimulated to get Th17 cells. It is usually to use 50 ng of PMA and 1 ug of Ionomicin for at least 5 hours in the presence of brefeldin A or monensin. Probably you could show 2-8% of Th17 cells.
Here is a publication of your interest
I hope this help you
Unless you isolate your naive T cells and differentiate them into Th17s you will get very little expression (Luis is correct around 2-8%, but it does vary from donor to donor).
In my experience, IL17+ cells after overnight incubation of PBMC with PMA/Iono + BFA are rather closer to 1-2% of CD4+ T cells than 2-8%...Incubation times are certainly an important variable to consider and test.
Rather than using brefeldin A also add monensin as you get better staining if you use both to block protein transport. I would recommend the protein transport inhibitor from eBiosciences.
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