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 . .
