Reagent List

  • Sterile PBS
  • Cell culture medium (IMDM supplemented with 10% FBS)
  • Sterile plastic petri dishes
  • RBC Lysis Buffer (Cat. No. 420301)
  • Anti-mouse CD3ε, clone 145-2C11 (Ultra-LEAF™ format, Cat. No. 100339)
  • Anti-mouse CD28, clone 37.51 (Ultra-LEAF™ format, Cat. No. 102116)
  • Anti-mouse IFN-γ, clone XMG1.2, (Ultra-LEAF™ format, Cat. No. 505834)
  • Mouse MojoSort™ CD4 T-cell Isolation Kit (Cat. No. 480005)
  • Anti-mouse IL-4, clone 11B11, (Ultra-LEAF™ format, Cat. No. 504122)
  • Recombinant mouse IL-6 (carrier-free) (Cat. No. 575704)
  • Recombinant mouse IL-23 (carrier-free) (Cat. No. 589002)
  • Recombinant human TGF-β1 (carrier-free) (Cat. No. 580702)
  • Brefeldin A (Cat. No. 420601)
  • Monensin Solution (Cat. No. 420701)
  • PMA (Phorbol 12-myristate 13-acetate) (Cat. No. P8139 from Sigma)
  • Ionomycin (Cat. No. I0634 from Sigma)

 

Protocol Steps

Isolation of CD4+ Cells From Lymph Nodes:

 

  1. Harvest lymph nodes (superficial cervical, mandibular, axillary, inguinal, and mesenteric) from mice.  

  2. Tease lymph nodes through a sterile 70-µm nylon cell strainer to obtain single-cell suspensions in complete IMDM containing 10% FCS (complete medium).  

  3. 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:

 

 

  1. 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.

  2. 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).  

  3. On day 3, slowly add 5ml of fresh media along with same the concentration of antibodies/cytokines as used on day 0.  

  4. 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.

  5. After harvesting, the cells are ready for staining.
    Tip: Recombinant human TGF-β is effective for stimulating mouse cells.