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.