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

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:

 

  1. 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.

  2. 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).  

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

  4. 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.

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