2.5 Flow cytometry analysis of Th2 cells
To measure cytoplasmic expression of markers by Th2 cells, cells cultured in a 37◦C/5% CO2 incubator were stimulated for 24 h with ionomycin (250 ng/ml; SigmaAldrich), PMA (25 ng/ml; Sigma-Aldrich), and monensin (20 ng/mL; eBio-science, San Diego, CA, USA). The cells were then stained for surface markers,
fixed, and permeabilized using the Cytofix/Cytoperm kit (eBioscience), and then stained with antibodies specific for intracellular cytokines or with corresponding isotype-matched controls, followed by analysis using a FACS Canto II flow cytometer (BD Biosciences, Mississauga, ON, Canada). The vitality of cells was discriminated using fixable viability stain 450 (FVS450, BD Biosciences). Validated antibodies (CD3-eFour450A、CD4-FITC、IL-4-PE) were purchased from eBioscience.
2.6 CBA detection of plasma IgE, IL-4 and IL-13
The plasma levels of IgE, IL-4 and IL-13 were measured using a CBA kit (eBioscience) according to the manufacturer’s instructions. All samples were measured in duplicate.