In vitro differentiation of Th cells from naïve CD4+ T cells is achieved by stimulating their T cell receptor (TCR) in the presence of appropriate cytokines. Here we describe a comprehensive protocol for in vitro differentiation of Th0, Th1, Th2, Th17 and iTreg T cells from mouse CD4+ T cells.
Day 1: Coat wells with anti-CD3 and CD28 antibody
1. Prepare anti-CD3 and anti-CD28 antibodies at 1μg/mL in sterile PBS.
2. Add 250~500 μL total volume per well to 24-well plates.
3. Cover and wrap in parafilm to prevent evaporation and contamination, then incubated at 4 °C.
Note: Concentration of anti-CD3 at 2 μg/ml and anti-CD28 at 1 μg/ml is optimal for Th17 generation.
Day 2: Differentiation of Th subtypes
4. Aspirate anti-CD3 and anti-CD28 antibodies solution from the wells of the coated plates, wash each well with 1 mL of sterile PBS.
5. Prepare differentiating culture medium:
6. Dissolve 1 × 106 naïve CD4+ T cells with 500 μL of differentiating culture medium.
Learn more about naïve CD4+ T cells isolation
7. Plate cells in each well of anti-CD3 and anti-CD28 coated 24-well plate.
8. Incubate cells in 5 % CO2 incubator at 37 °C for 4~5 days.
Day 7: Analysis
9. Generally, differentiated Th cells are analyzed via cytokines detection and/or transcription factors identification. IFN-γ, IL-4, IL-17, and Foxp3 are typically used markers for Th1, Th2, Th17, and iTreg differentiations, respectively. The following approaches are alternative for Th subsets analysis: