Th1 Polarization of Mouse CD4+ Cells Protocol
Isolation of CD4+ Cells From Lymph Nodes:
- 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 in complete RPMI containing 10% FCS (complete medium).
- Resuspend cells in complete medium and use your favorite method to isolate CD4+ cells. Consider using our Mojosort™ Mouse CD4 T Cell Isolation Kit.
Th1 Polarization of CD4+ Cells:
- On day 0, coat 12-well plate with anti-mouse CD3ε, clone 145-2C11 (3µg/ml). Incubate at 37°C for 2 hours or 4°C overnight. Aseptically decant antibody solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
- Plate CD4+ cells at 1.0 x 106/1ml/well. Culture cells for 5 days at 37°C, 5% CO2, in the presence of anti-mouse CD28, clone 37.51 (3µg/mL), anti-mouse IL-4, clone 11B11 (10µg/mL), recombinant mouse IL-2 (5ng/mL), and recombinant mouse IL-12 (10ng/ml).
- On day 3, if media is yellow, add 2ml/well of fresh media.
- On day 5, wash cells once and then restimulate in complete media with 50ng/ml PMA, 1µg/ml ionomycin and 10µl monensin (1000x), in a 6-well plate in incubator at 37°C for 5 hours.
- After harvesting, the cells are ready for staining.