Th17 Polarization of Mouse CD4 Cells
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实验试剂
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.
