Th2 Polarization of Mouse CD4+ Cells Protocol

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

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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.

 

Th2 Polarization of CD4⁺ Cells:

 

 

4. 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).


5. 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).


6. 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.


7. 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.


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