Treg 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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Plate CD4 + cells at 1.0 x 106 / 1ml / well .
Culture cells for 5 days at 37°C , 5 % CO2 , in the presence ofanti - mouse CD28 , clone 37.51 ( 3 µg / mL ) , recombinant mouse IL - 2 ( 5 ng / mL ) , and recombinanthuman TGF - β1 ( 5 ng / ml ) .
On day 3 , if media is yellow , add 2 ml / well of fresh media .
On day 5 , after harvesting , the cells are ready for staining .
On day 0 , coat 12 - well plate with anti - mouse CD3ε , clone 145 - 2C11 ( 3 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 .
