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.
