In vitro Differentiation of Mouse Th0, Th1 and Th2 from Naïve CD4 T Cells   

Abstract

Materials and Reagents

1. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 900-108 )
   
2. Penicillin/streptomycin solution (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
3. 2-mercaptoethanol (Life Technologies, Invitrogen^TM, catalog number: 21985-023 )
   
4. IL-2 (R&D systems, catalog number: 402-ML-020 )
   
5. IL-4 (Biolegend, catalog number: 574302 )
   
6. IL-12 (R&D systems, catalog number: 419-ML-010 )
   
7. Anti-CD3 (Biolegend, catalog number: 100302 )
   
8. Anti-CD28 (Biolegend, catalog number: 102102 )
   
9. Anti-IL-4 (Biolegend, catalog number: 504102 )
   
10. Anti-IL-12 (Biolegend, catalog number: 505303 )
    
11. Anti-IFNg (Biolegend, catalog number: 505702 )
    
12. Phorbol 12-myristate-13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
    
13. Ionomycin (Sigma-Aldrich, catalog number: 19657 )
    
14. Monensin (ebioscience, catalog number: 00-4505-51 )
    
15. Easysep CD4⁺ T cell enrichment Kit (STEMCELL Technologies, catalog number: 19752 )
    
16. RPMI-1640 medium (Life Technologies, Gibco^®, catalog number: 11875-093 ) (see Recipes)
    

Equipment

1. 48, 96-Well plate
   
2. Centrifuges
   
3. Water bath
   
4. Fluorescence activated cell sortor (FACS)
   

Procedure

1. Enrich CD4⁺ T cells by Easysep CD4⁺ T cell enrichment kit following the manufacturer’s instructions. Make sure the purity of CD4⁺ T cells is above 70% after enrichment.
   
2. Sort CD4⁺ CD44loCD62Lhi naïve T cells from enriched CD4⁺ T cells.
   
3. Count the sorted cells and plate them as follows:
   
   1. For 96-well-plate, 1 x 10⁵ cells/well in 200 μl culture medium for each well.
      
   2. For 48-well-plate, 2 x 10⁵ cells/well in 500 μl culture medium for each well.
4. Set up the differentiation culture (numbers are final concentration)
   

   	Th0	Th1	Th2	Th17
   anti-CD3 (plate coated)	5 μg/μl	5 μg/μl	5 μg/μl	5 μg/μl
   anti-CD28	1 μg/ml	1 μg/ml	1 μg/ml	1 μg/ml
   IL-2	20 ng/ml	20 ng/ml	20 ng/ml	20 ng/ml
   IL-4			100 ng/ml
   IL-12		20 ng/ml
   anti-IL-4		10 ng/ml		10 ng/μl
   anti-IFNg			10 ng/ml	10 ng/ml
   anti-IL-12			10 ng/ml

   
   
5. Transfer the culture to a new plate after 2 days without digesting or changing the medium.
   
6. Culture for 3 more days. If medium turns yellow, replace it with new medium with neutralizing antibodies (no cytokines).
   
7. Add 50 ng/ml PMA and 1 μg/ml ionomycin to the cells and incubate for 1 h.
   
8. Add 2 μM monensin and incubate cells for another 4 h.
   
9. Verify the lineage of T helper cells by determine the levels of intracellular IFN-gamma and IL-4 by FACS.
   Note: Please see the following link for common FACS staining protocol (https://bio-protocol.org/e31).
   

Recipes

1. RPMI-1640 culture medium
   Supplemented with
   5% FBS
   1% Penicillin/streptomycin solution
   50 μM 2-mercaptoethanol