This protocol describes the isolation of naive human T cells and culture conditions that result in the development of IL-17 producing helper Th17 cells. The addition of IL-23 or IL-1 is essential for this process as is T cell receptor activation.
Method Article
In vitro human Th17 development
https://doi.org/10.1038/nprot.2007.345
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This protocol describes the isolation of naive human T cells and culture conditions that result in the development of IL-17 producing helper Th17 cells. The addition of IL-23 or IL-1 is essential for this process as is T cell receptor activation.
-Recombinant hIL-1β (50 ng/ml), rhIL-4 (10 ng/ml), rhIL-12 (5 ng/ml), rhIL-6 (30 ng/ml), rhTGFβ (10 ng/ml), and rhIL-2 (100 U/ml) were purchased from R&D Systems. Recombinant hIL-23 was generated in house.
-Medium : Yssel’s medium with 1% human serum AB was from Gemini Bio-Products
-Human CD4+ T cell isolation kit II (#130-091-155), human CD45RO microbeads (#130-046-001), and human T cell activation/expansion kit (#130-091-441) were from Miltenyi Biotec.
AutoMACS, Milteniy Biotec
-. Subsequently, 30 µl of MACS buffer per 10 cells was added, and cells were incubated with 20 µl of Anti-Biotin Microbeads per 10 cells for 15 min at 4°C.
Cells were washed with MACS buffer and resuspended up to 10 cells in 500µl of MACS buffer.
CD4+ T cells were sorted by two rounds of magnetic depletion on the AutoMACS (program Deplete S).
14 days
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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