Reagent List

  • Sterile PBS
  • Cell culture medium (RPMI 1640 supplemented with 10% FBS)
  • Sterile plastic petri dishes
  • Sterile T-75 culture flask
  • RBC Lysis Buffer (Cat. No. 420301)
  • Concanavalin A (Con A) (Sigma, Cat. No. C5275)
  • 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 mouse IL-2 (carrier-free) (Cat. No. 575402)
  • Recombinant mouse IL-4 (carrier-free) (Cat. No. 574302)
  • Monensin Solution (Cat. No. 420701)
  • 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.

 

Th2 Polarization of CD4+ Cells:

 

 

  1. On day 0, plate CD4+ cells at 30 x106/30 ml/T-75 flask. Culture cells for 3 days in complete RPMI containing 10% FCS, Con A (5µg/mL), recombinant mouse IL-2 (20ng/ml), and recombinant mouse IL-4 (50ng/ml).

  2. On day 3, harvest the cells and wash cells once. Seed 15 x106 cells/30 ml/T-75 flask along with recombinant mouse IL-2 (20ng/ml) and recombinant mouse IL-4 (50ng/ml).

  3. On day 5, coat a 60 x 15 mm tissue culture petri dish with anti-mouse CD3ε, clone 145-2C11, 10µg/mL in PBS, 5ml/dish. Incubate in a 4°C refrigerator overnight.

  4. On day 6, wash the anti-mouse CD3ε pre-coated tissue culture petri dish with PBS. Harvest the cells from the flask (that were seeded on Day 5), wash them twice, and seed at 20 x106/10 ml/petri dish along with 10µl of monensin (1000x) and anti-mouse CD28, clone 37.51 (5µg/ml). Incubate for 6 hours at 37°C in a CO2 incubator.

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