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 .
