Th17 Polarization of Mouse CD4+ Cells
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 IMDM 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.
Th17 Polarization of CD4+ Cells:
- On day 0, coat 60 x 15mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11 (5µg/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 10 x 106/5 ml/dish. Culture cells for 4 days in the presence of anti-mouse CD28, clone 37.51 (5µg/mL), recombinant mouse IL-6 (50ng/mL), recombinant human TGF-β1 (1ng/mL), recombinant mouse IL-23 (5ng/ml), anti-mouse IL-4 (10µg/mL), and anti-mouse IFN-γ (10µg/mL).
- On day 3, slowly add 5ml of fresh media along with same the concentration of antibodies/cytokines as used on day 0.
- On day 4, wash cells once and then restimulate in complete medium with 500ng/ml PMA and 500ng/mL ionomycin, in the presence of Brefeldin A (If you are looking for IL-21 production, use monensin) for 4-5 hours.
- After harvesting, the cells are ready for staining.
Tip: Recombinant human TGF-β is effective for stimulating mouse cells.