Th17 Polarization of Mouse CD4⁺ Cells

 

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. 781802) 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

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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:

 

 

4. 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.


5. 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).  


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


7. 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.


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