Th9 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 in IMDM 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 or 4˚C overnight.
Aseptically decant antibody solution from the plate.
Wash plate 3 times with sterile PBS.
Discard liquid.
Plate CD4+ cells at 10 x106/5 ml/dish.
Culture cells for 3 days in the presence of anti-mouse CD28, clone 37.51 (5 μg/mL), recombinant human TGF-β1 (10 ng/mL), recombinant mouse IL-4(10 ng/mL), recombinant mouse IL-2 (20 ng/ml), and anti-mouse IFN-γ, clone XMG1.2 (10 μg/mL).
On day 3, wash cells once and then restimulate in complete medium with 500 ng/ml PMA and 500 ng/mL ionomycin, in the presence of monensin for 6 hours.
After harvesting, the cells are ready for staining..
