I am trying to expand TH17 cells from Naive T cells for the first time. Can you suggest the best expansion kit available in the market to do the same?
Hello Ramya Parandaman
IL6, IL21, IL23, and TGF-β are the major signaling cytokines involved in Th17 cells differentiation, and retinoic acid receptor-related orphan receptor gamma-T (RORγt) is the master regulator. The differentiation process can be split into 3 stages,
The differentiation stage mediated by TGF-β and IL6,
The self-amplification stage mediated by IL21, and
The stabilization stage mediated by IL23.
If you are interested in mouse, then you may use the kit attached below. The Flow Cellect Mouse Th17 Differentiation Tool Kit Catalog No. FCIM025163 from Millipore.
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/389/881/fcim025163-rev-f.pdf
For human, RnD systems used to provide the Human Th17 Cell Differentiation Kit which has been discontinued. See below.
https://www.rndsystems.com/products/cellxvivo-human-th17-cell-differentiation-kit_cdk003
https://www.rndsystems.com/products/cellxvivo-human-th17-cell-differentiation-kit_cdk003b
https://www.rndsystems.com/products/cellxvivo-human-th17-cell-differentiation-kit_cdk003c
Nevertheless, you could follow the protocol provided below for human Th17 cell differentiation.
1. You may purify naive CD4+CD45RA+ T cells from PBMCs using the Naive T Cell Isolation Kit (human).
2. You may stimulate the cells by using the T Cell Activation/ Expansion Kit, (human) which is based on MACSi bead particles loaded with the required antibodies.
3. You may culture the cells in the presence of the Th17-polarizing cytokines, like IL-1β (20 ng/mL), IL-6 (30 ng/mL), IL-23 (30 ng/mL), and TGF-β1 (2.25 ng/mL), in addition to anti-IFN-γ (1 µg/mL) and anti-IL-4 (2.5 µg/mL) antibodies. Culture the cells for 7 days at 37 °C in an atmosphere of 5% CO2, without any media exchange.
4. You may collect the cell culture supernatants, filter, and measure the content of IFN-γ, IL-4, IL-17, IL-22, by ELISA method.
For detailed protocol you may want to refer to the link below.
https://www.miltenyibiotec.com/_Resources/Persistent/b17b9473ea04defee43a0afedf572d45ead420c8/Th17%20cell%20differentiation%20%28customer%20report%29.pdf
Best.
Hello Malcolm Nobre
Thank you so much for the detailed explanation and protocols attached. I will follow the steps accordingly!
Regards,
Ramya
Malcolm Nobre
Hi, you mentioned about differentiating Naive CD4+ T cells to TH17 using Th17 specific antibodies without changing the media.
I am working on developing the same protocol, however, I noticed that the media changes yellow on day 4 and also noticed that the viability is decreasing. Is it okay to leave it yellow? Do you have any suggestions?
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