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.
