Th2 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 , plate CD4 + cells at 30 x106 / 30 ml / T - 75 flask .
Culture cells for 3 days in complete RPMI containing 10 % FCS , Con A ( 5 µg / mL ) , recombinant mouse IL - 2 ( 20 ng / ml ) , and recombinant mouse IL - 4 ( 50 ng / ml ) .
On day 3 , harvest the cells and wash cells once .
Seed 15 x106 cells / 30 ml / T - 75 flask along with recombinant mouse IL - 2 ( 20 ng / ml ) and recombinant mouse IL - 4 ( 50 ng / ml ) .
On day 5 , coat a 60 x 15 mm tissue culture petri dish with anti - mouse CD3ε , clone 145 - 2C11 , 10 µg / mL in PBS , 5 ml / dish .
Incubate in a 4°C refrigerator overnight .
On day 6 , wash the anti - mouse CD3ε pre - coated tissue culture petri dish with PBS .
Harvest the cells from the flask ( that were seeded on Day 5 ) .
Wash the cells .
( wash 1 / 2 ) Wash the cells .
( wash 2 / 2 ) Seed at 20 x106 / 10 ml / petri dish along with 10 µl of monensin ( 1000x ) and anti - mouse CD28 , clone 37.51 ( 5 µg / ml ) .
Incubate for 6 hours at 37°C in a CO2 incubator .
After harvesting , the cells are ready for staining .
