Treg Polarization of Mouse CD4+ Cells

 

Reagent List

  • Sterile PBS
  • Cell culture medium (RPMI 1640 supplemented with 10% FBS)
  • Sterile 12-well plate
  • 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)
  • Recombinant human TGF-β1 (carrier-free) (Cat. No. 580702)
  • Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402)
  • Mouse MojoSort™ CD4 T-cell Isolation Kit (Cat. No. 480005)

 

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.  

Treg 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),recombinant mouse IL-2 (5ng/mL), and recombinant human TGF-β1 (5ng/ml).  

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

  4. On day 5, after harvesting, the cells are ready for staining.
    Note: Recombinant human TGF-β is effective for stimulating mouse cells.  
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