Journal: Frontiers in Immunology
Article Title: Acidic Microenvironment Regulates the Severity of Hepatic Ischemia/Reperfusion Injury by Modulating the Generation and Function of Tregs via the PI3K-mTOR Pathway
Figure Lengend Snippet: Reversal of acidic microenvironment restores Foxp3 expression and iTreg function. (A) The proportion of CD4 + CD25 + Foxp3 + iTregs cultured with IL-2 and TGF-β for 3 days. (B,C) The proportion of CD4 + IL-10 + and CD4 + TGF-β + T cells cultured with IL-2 and TGF-β for 3 days and stimulated with phorbol 12-myristate 13-acetate, brefeldin A, and ionomycin for 6 h before the assay. (D) Statistical analysis of the suppression assay in vitro . (E) iTregs induced in media of varying pH values for 3 days were co-cultured with carboxyfluorescein succinimidyl ester-stained human naïve CD4 + T cells (responders) at the indicated ratio. After 72 h of activation with anti-CD3/CD28-conjugated beads, responder cell proliferation was assessed by flow cytometry. (F–H) Statistical analysis of related results of flow cytometry. Data are presented as the means ± SD from three independent experiments. *** p
Article Snippet: For intracellular staining of cytokines, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml, Biogems), ionomycin calcium salt (1 μg/ml, Biogems), and brefeldin A (5 μg/ml, Biogems) for 6 h. Then, cells were stained with surface markers and further fixed/permeabilized (BioLegend) and stained for intracellular protein.
Techniques: Expressing, Cell Culture, Suppression Assay, In Vitro, Staining, Activation Assay, Flow Cytometry