Figure 5.

Esculetin also induces CD4⁺Foxp3⁺ Tregs in vitro. CD4⁺CD25⁻ T cells were isolated from naïve BALB/c mice via cell sorting by FACSAria III and then co-stimulated with anti-CD3 (5 μg/ml) plus anti-CD28 mAb (2.5 μg/ml) in the presence or absence of IL-2 (10 ng/ml) plus esculetin (10 μM) or TGF-β1 (5 ng/ml) for 4 days. CD4⁺Foxp3⁺ Treg frequencies were determined by flow cytometric analysis (A). Data of column graphs are presented as the mean ± SD (n = 4–6, *p < 0.05 and **p < 0.01). One representative of three separate experiments is shown (B).