Anti il 9 pe

The splenic cells were gently dispersed through nylon mesh into a single-cell suspension, washed with RPMI 1640 (Gibco, USA). Single cell suspensions were prepared by lysing red blood cells with red blood cell lysing buffer (BD Biosciences, Vienna, Austria). The splenic mononuclear cells were then resuspended in RPMI 1640 medium with 10% FCS (Gibco, USA), stimulated with phorbol myristate acetate (PMA, 25 ng/ml, Sigma-Aldrich Poole, UK) and ionomycin (1 μg/ml, Sigma-Aldrich) in the presence of GolgiPlug (1 μl/10⁶cells, BD Biosciences) on a 24-well culture plate at 37 °C. After 5 h incubation, the cells were harvested and stained with PerCP-Cy5.5 conjugated anti-mouse CD4 antibody (BD Biosciences). Then cells were stained intracellularly with anti-IL-9-PE (eBioscience, San Diego, CA, USA), anti-IL-17-PE (eBioscience, USA), PE-conjugated anti-IL22 mouse antibody (eBioscience, USA) and anti-IFN-γ-Alexa-Fluor®488 (BD Biosciences) mouse antibody after fixation and permeabilization (BD Biosciences).

PBMCs or IHLs were stimulated with either phorbol myristate acetate (PMA) (50 ng/ml)+ionomycin (1 μg/mL) or HBsAg (10 μg/mL) in the presence of brefeldin A (10 μg/mL) for 6 hours. Cells were transferred to FACS tubes, and were stained with anti-CD3-FITC (BD Bioscience, San Jose, CA, USA), anti-CD4-PerCP (BD Bioscience), and anti-CD8-APC (BD Bioscience) for 30 min in the dark at 4 °C. Cells were washed twice, and were stained with anti-IL-9-PE (eBioscience, San Diego, CA, USA) for 30 min at room temperature after fixation and permeabilization. Isotype controls were used to enable correct compensation and confirm antibody specificity. Acquisitions were performed using Cell Quest Pro Software (BD Bioscience Immunocytometry Systems, San Jose, CA, USA) in a FACS Calibur analyzer (BD Bioscience Immunocytometry Systems). Data were analyzed using FlowJo Software Version 10.0 for Windows (Tree Star, Ashland, OR, USA).