Th17 Polarization of Mouse CD4+ Cells
Harvest lymph nodes (superficial cervical, mandibular, axillary, inguinal, and mesenteric) from mice.
Tease lymph nodes through a sterile 70-µm nylon cell strainer to obtain single-cell suspensions incomplete RPMI containing 10% FCS (complete medium).
Resuspend cells in complete medium and use your favorite method to isolate CD4+ cells.
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On day 0, coat 60 × 15 mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11 (5 µg/ml).
Incubate at 37°C for 2 hours.
(Alternatively, incubate at 4°C overnight.)
Aseptically decant antibody solution from the plate.
Wash plate with sterile PBS (wash 1/3).
Wash plate with sterile PBS (wash 2/3).
Wash plate with sterile PBS (wash 3/3).
Discard liquid.
Plate CD4+ cells at 1.0 x 106 /1ml/well.
Culture cells for 4 days in the presence of anti-mouse CD28, clone 37.51 (5 µg/mL), recombinant mouse IL-6 (50 ng/mL), recombinant human TGF-β1 (1 ng/mL), recombinant mouse IL-23 (5 ng/ml), anti-mouse IL-4 (10 µg/mL), and anti-mouse IFN-γ (10 µg/mL).
On day 3, slowly add 5 ml of fresh media along with same the concentration of antibodies/cytokines as used on day 0.
On day 4, wash cells once and then restimulate in complete medium with 500 ng/ml PMA and 500 ng/mL ionomycin, in the presence of Brefeldin A (If you are looking for IL-21 production, use monensin) for 4 - 5 hours.
After harvesting, the cells are ready for staining.
