Naïve CD4 T cells (CD4+TCR-Vβ11+CD25-CD62LhighCD44low) were isolated from splenocytes of 8- to 10-week-old 2D2 mice using the CD4 T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions, followed by FACS sorting. The antigen-presenting cells were irradiated with a 3500 rad gamma ray and seeded with the sorted naïve CD4 T cells at a 5:1 ratio. T cells were activated using 40 μg/ml of MOG35-55 (AnyGen, Korea), and differentiated using the following cytokine cocktails in a 96-well flat bottom plate for 3 days: 5 μg/ml of anti-IL-4 (11B11; 16-7041-85; eBioscience), 5 μg/ml of anti-IFN-γ (XMG1.2; 16-7311-85; eBioscience), 30 ng/ml of IL-6 (406-ML; R&D Systems), 2 ng/ml of TGF-β (240-B-002; R&D Systems) for Th17 differentiation; or 5 ng/ml of TGF-β for Treg differentiation. Additionally, cells were co-treated with dNP2-ctCTLA-4 or CTLA-4-Ig.
Il 6 406 ml
Manufactured by R&D Systems
IL-6 (406-ML) is a recombinant human interleukin-6 protein. Interleukin-6 is a cytokine involved in various biological processes, including the immune response, inflammation, and hematopoiesis.
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2 protocols using il 6 406 ml
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Naïve CD4 T Cell Differentiation
2
Recombinant Proteins and Inhibitor Reagents for Cell Signaling
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Recombinant proteins were purchased from R&D Systems: EDA-A1 (3944-ED), EDA-A2 (922-ED), TNFα (410-MT), BAFF (8876-BF), TWEAK (1237-TW), OSM (495-MO), IL-6 (406-ML) and LIF (8878-LF). Small molecule inhibitors were acquired from indicated sources: TPCA-1 (abcam, ab145522), MG132 (Sigma; M8699), BAY 11-7082 (abcam, ab141228), BOT-64 (Santa Cruz; sc-222062), B022 (Aobious; AOB8699). Mouse EDA-A1 (MC208411), mouse EDA-A2 (MC208415) and mouse OSM (MR226014) expression plasmids were purchased from Origene.
Mouse NIK expression plasmid was purchased from Invivogen (pUNO1-mMap3k14). Wild-type human NIK and the NIK-K429/430A mutant plasmids were a gift from Prof. Michael Kracht (JLU Giessen).
Mouse NIK expression plasmid was purchased from Invivogen (pUNO1-mMap3k14). Wild-type human NIK and the NIK-K429/430A mutant plasmids were a gift from Prof. Michael Kracht (JLU Giessen).
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