Interleukin 6 (il 6)

Spleen cells were excised from mice and single-cell suspensions of splenocytes were prepared in RPMI 1640 by teasing the organ through a sterile nylon mesh. Naive CD4⁺ T cells were purified using an EasySep mouse naive CD4⁺ T cell isolation kit (Stemcell Technologies, Vancouver, BC, Canada) and cultured with plate-bound anti-CD3 (5 µg/ml; Bio X Cell), anti-CD28 (5 µg/ml; Bio X Cell), and cytokines used alone, including IL-12 (R&D Systems), IFN-γ (R&D Systems), IL-1 (R&D Systems), IL-4 (R&D Systems), IL-6 (Cell Signaling Technology), IL-23 (eBioscience), and TGF-β (Invitrogen), or in combination with Abs (Bio X Cell) for Th17-promoting (2.5 ng/ml TGF-β, 20 ng/ml IL-6, 10 µg/ml anti–IFN-γ, 10 µg/ml anti–IL-4, 10 µg/ml anti–IL-2), Th1-promoting (10 ng/ml IL-12, 5 µg/ml anti–IL-4), Th2-promoting (10 ng/ml IL-4, 5 µg/ml anti–IFN-γ), or regulatory T cell (Treg)–promoting (5 ng/ml TGF-β, 5 µg/ml anti–IFN-γ, 5 µg/ml anti–IL-4) conditions. In some experiments, naive CD4⁺ T cells were cultured in Th17-promoting conditions and IL-21 (20 ng/ml; eBioscience) or IL-23 (50 ng/ml) was added to cultures after 24 h. Cells were harvested after 48 h for real-time PCR analysis and after 72 h for cytokine expression by flow cytometry as previously described (23 (link)).