Th17 Polarization of Mouse CD4 Cells
实验概要
T helper 17 (Th17) cells are a subset of CD4 T helper cells characterized by their production of IL-17, particularly IL-17A and IL-17F. They are involved in autoimmune diseases such as multiple sclerosis, psoriasis, autoimmune uveitis, rheumatoid arthritis, and juvenile diabetes, but are also important in anti-microbial defenses at epithelial/ mucosal barriers, particularly against Candida and Staphylococcus. In addition to IL-17, Th17 cells also produce IL-21 and IL-22 that contribute to pro-inflammatory responses. The transcription factors RORα and RORγ have been identified as important factors in the differentiation of these cells. Th17 cells can be induced in vitro by a variety of cytokines including TGF-β, IL-6, IL-21 and IL-23. Here we provide a effective protocol for the generation of mouse IL-17 producing cells in vitro.
主要试剂
Sterile PBS
Cell culture medium
Sterile plastic petridishes
RBC Lysis Buffer [Details: Biolengend, cat.#420301]
Anti-mouse CD3ε, clone145-2C11[Details: LEAF™format, cat.#100314]
Anti-mouse CD28, clone37.51[Details: LEAF™format, cat.#102112]
Anti-mouse IL-4 [Details: LEAF™format, cat#504108]
Anti-mouse IFN-γ [Details: LEAF™format, cat.#505812]
Recombinant mouse IL-6(carrier-free)[Details: Biolengend, cat.#575702]
Recombinant human TGF-β1(carrier-free)[Details: Biolengend, cat.#580702]
Recombinant mouse IL-23 (carrier-free)[Details: Biolengend, cat.#589002]
Brefeldin A[Details: Biolengend, cat.#420601]
PdBu(Phorbol12,13-dibutyrate)[Details: Sigma, cat.#P1269]
FICZ (6-formylindolo[3,2-b]carbazole)[Details: Life Sciences, cat. #BML-GR206-0100]
实验步骤
1. Isolation of CD4 cells from lymph nodes
1) Harvest lymph nodes (superfical cervical, madibular, axillary, inguinal, and mesenteric) from mice.
2) Tease lymph nodes through a sterile 70-µm nylon cell strainer to achieve single-cell suspensions in RPMI 1640 contain 10% FCS ( complete medium).
3) Resuspend cells in complete medium and use your favorite method to isolate CD4 cells. Check out Biocompare.com to find useful kits.
2. Th17 Polarization of CD4 cells
1) Coat 60×15 mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11(2μg/ml). Incubate at 37 °C for 2 hours or 4 °C over night. A septically decant antibody solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
2) Plate CD4 cells at 1x106/ml. Culture cells for 3 days in presence of anti-mouse CD28, clone 37.51(5μg/mL),IL-6 (50ng/mL),TGF-β1 (1ng/mL), anti-mouse IL-4 (10μg/mL), anti-mouse IFN-γ (10μg/mL), and 300 nM of FICZ. Alternatively, mouse IL-23 (5ng/ ml) can be used in place of FICZ.
3) On day 3, wash cells once and then restimulate in complete medium with 500ng/ml PdBU, and 500 ng/mL ionomycin, in the presence of Brefeldin A for 4-5 hours.
4) After harvesting, the cells are ready for staining.
