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In vitro Differentiation of Mouse Th0, Th1 and Th2 from Naïve CD4 T Cells
Published: Nov 20, 2011 DOI: 10.21769/BioProtoc.157 Views: 56513
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Abstract
In vitro differentiation of helper T cells of various lineages is frequently used in T helper cell study. Naïve CD4 T cells can differentiate into certain lineage of T help cells in vitro in the presence of specific stimulatory cytokines and inhibition of cytokines that are essential for the differentiation of other lineages.
Materials and Reagents
- Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 900-108 )
- Penicillin/streptomycin solution (Life Technologies, Gibco®, catalog number: 15140-122 )
- 2-mercaptoethanol (Life Technologies, InvitrogenTM, catalog number: 21985-023 )
- IL-2 (R&D systems, catalog number: 402-ML-020 )
- IL-4 (Biolegend, catalog number: 574302 )
- IL-12 (R&D systems, catalog number: 419-ML-010 )
- Anti-CD3 (Biolegend, catalog number: 100302 )
- Anti-CD28 (Biolegend, catalog number: 102102 )
- Anti-IL-4 (Biolegend, catalog number: 504102 )
- Anti-IL-12 (Biolegend, catalog number: 505303 )
- Anti-IFNg (Biolegend, catalog number: 505702 )
- Phorbol 12-myristate-13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
- Ionomycin (Sigma-Aldrich, catalog number: 19657 )
- Monensin (ebioscience, catalog number: 00-4505-51 )
- Easysep CD4+ T cell enrichment Kit (STEMCELL Technologies, catalog number: 19752 )
- RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-093 ) (see Recipes)
Equipment
- 48, 96-Well plate
- Centrifuges
- Water bath
- Fluorescence activated cell sortor (FACS)
Procedure
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Immunology > Immune cell function > Lymphocyte
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