I’m currently trying to differentiate Th17 cells from purified CD4+ T cells isolated from human PBMCs. My current protocol involves:
- Activation: 1 µg/mL anti-CD3 and 1 µg/mL anti-CD28
- Polarizing conditions: Recombinant TGF-β1: 2.5 ng/mL Recombinant IL-6: 20 ng/mL Anti-IFN-γ: 1 µg/mL Anti-IL-4: 1 µg/mL
- Culture medium: RPMI-1640 supplemented with 10% FBS and standard additives
- Duration: 5 days
On day 5, I treat the cells with Brefeldin A (10 µg/mL for 3 hours), then stain for CD4 and IL-17A to assess the frequency of CD4⁺IL-17⁺ cells by flow cytometry.
However, I am consistently observing very low induction of IL-17A⁺ cells. Could anyone suggest me any improvements in my experimental setup for optimizing Th17 differentiation under these conditions?
Here's a protocol I've had success with.
Reagents
- anti-CD3 - 2μg/mL
- anti-CD28 - 5μg/mL
Th17 polarization medium:
- Advanced RPMI
- 10% heat inactivated FBS
- 1:100 GlutaMax
- 1:100 Pen-strep
- IL-1b - 20ng/mL
- IL-6 - 40 ng/mL
- anti-IL-4 - 5μg/mL
- anti-IFNγ - 10μg/mL
Expansion medium
- Th17 medium + 10 units/mL of IL-2 (Roche) + IL-23 (20 ng/mL)
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Thank you for the response Lauren. I just want to know that you have not used recombinant TGF-beta. Does it hinders Th17 polarization?
Hi Najma,
I made a correction to my initial protocol, IL-23 is also included in the expansion medium.
Our goal was a more pathogenic phenotype of Th17 so we omitted the TGF-beta, however adding TGF-beta might make it more efficient.
I didn't observe any hindrance, I was able to polarize cells in large enough quantities sufficient for my needs. You might want to play around with TGF-beta concentrations to see what is optimal for your purposes.
Here is some literature that might help with the optimization.
https://academic.oup.com/jimmunol/article/187/2/692/7973878?login=true#508812544
https://pmc.ncbi.nlm.nih.gov/articles/PMC7581618/#S9
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