Pe conjugated anti mouse il 4

Following treatment of purified CD4+ T cells with different drugs, the concentrations of Th1 cells and Th2 cells were investigated by flow cytometry (BD Bioscience, USA). To facilitate the intracellular staining of T helper cells, all cells were stimulated with Cell Activation Cocktail (with Brefeldin A) (BioLegend, USA) for 5 hours before detection. Staining of T helper cells for flow cytometry requires two steps: extracellular staining and intracellular staining. After the harvested cells were washed with FACS buffer, FITC-conjugated anti-mouse CD4 (BioLegend, USA) was added to the cells for 30 minutes at RT. To decrease the nonspecific Fc receptor-mediated Ab staining, cells were preincubated with mouse IgG for 20 minutes at 4°C. Then, the FIX&PERM Kit (FMS, China) was used according to the manufacturer's instructions to fix and permeabilize the cells. APC-conjugated anti-mouse IFN-γ (BioLegend, USA) and PE-conjugated anti-mouse IL-4 (BioLegend, USA) were added to bind to intracellular cytokines. Th1 cells and Th2 cells were labeled with CD4 + IFN-γ and CD4 + IL-4, respectively.

Recombinant mouse IFN-γ, anti-mouse CD3e, recombinant mouse IL-2, FITC conjugated anti-mouse CD4, PE conjugated anti-mouse IFN-γ, granzyme B, perforin, PE-Cy5 conjugated anti-mouse CD8a, APC conjugated anti-mouse NK1.1 and isotype controls of each antibody were purchased from eBioscience. FITC conjugated anti-mouse CD3e, PE conjugated anti-mouse IL-4 and IL-5 and isotype controls of each antibody were purchased from BioLegend. R-PE labeled CD1d tetramer preloaded with α-GalCer and R-PE labeled CD1d tetramer negative control were purchased from Proimmune. The mouse IFN-γ, IL-4, IL-5 and IL-13 ELISA kits were purchased from eBioscience. Alum adjuvant was purchased from Thermo Fisher Scientific. Ovalbumin was purchased from Sigma-Aldrich. The endotoxin level in Ovalbumin is < 1 EU/mg.