In Vitro Differentiation of T Cells

1. In Vitro Differentiation of T Cells 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. Coat wells with anti-CD3 and CD28 antibody1. Prepare anti-CD3 and anti-CD28 antibodies at 1μg/mL in sterile PBS.Add 250~500 μL total volume per well to 24-well plates.Cover and wrap in parafilm to prevent evaporation and contamination, then incubated at 4 °C. Concentration of anti-CD3 at 2 μg/ml and anti-CD28 at 1 μg/ml is optimal for Th17 generation. Differentiation of Th subtypes. Aspirate anti-CD3 and anti-CD28 antibodies solution from the wells of the coated plates, wash each well with 1 mL of sterile PBS. Prepare differentiating culture medium: https://www.creative-diagnostics.com/in-vitro-differentiation-of-t-cells.htm