Th1 Polarization of Mouse CD4⁺ Cells Protocol

 

Reagent List

Sterile PBS Cell culture medium (RPMI 1640 supplemented with 10% FBS) Sterile 12-well plate Sterile 6-well plate RBC Lysis Buffer (Cat. No. 420301) Anti-mouse CD3ε, clone 145-2C11 (Ultra-LEAF™ format, Cat. No. 100339) Ionomycin (Cat. No. I0634 from Sigma) Mouse MojoSort™ CD4 T-cell Isolation Kit (Cat. No. 480005)

Anti-mouse CD28, clone 37.51, (Ultra-LEAF™ format, Cat. No. 102116) Anti-mouse IL-4, clone 11B11, (Ultra-LEAF™ format, Cat. No. 504122) Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402) Recombinant mouse IL-12 (p70) (carrier-free) (Cat. No. 577002) Monensin Solution (Cat. No. 420701) PMA (Phorbol 12-myristate 13-acetate) (Cat. No. P8139 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 RPMI 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.  

Th1 Polarization of CD4⁺ Cells:

 

4. 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.


5. 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).  


6. On day 3, if media is yellow, add 2ml/well of fresh media.  


7. 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.


8. After harvesting, the cells are ready for staining.