Il 5 apc

For cell culture and virus preparation, Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY, USA) and Lonza (Basel, Switzerland), respectively. To measure cytokine concentrations, cytometric bead array (CBA) mouse inflammation/Th1/Th2/Th17 cytokine kit, mouse IL-5 and IL-13 flex sets were purchased from BD Biosciences (San Diego, CA, USA). All reagents for flow cytometry including Golgi Plug, Cytofix/Cytoperm solution, anti-mouse CD16/32 (Mouse BD Fc Block^TM), CD4-APC-Cy7, CD44-FITC, IFN-γ-APC, IL-17-PE, IL-5-APC, CD11c-FITC, CD45-PerCP, Siglec-F-PE, and Ly6G-PE-Cy7 were purchased from BD Biosciences. For development of ELISA, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Southern Biotechnology (Birmingham, AL, USA). HRP-conjugated goat anti-mouse IgG1 and IgG2a were purchased from Zymed Laboratories (San Francisco, CA, USA).

The cells were thawed and washed with PBS first and PBS/1% BSA later and then stained with surface antibodies for 30–60 minutes. Surface antibodies used were CD3 - Amcyan, CD4 - APC-H7 and CD8 - PE-Cy7 (all from BD Biosciences). The cells were washed and permeabilized with BD Perm/Wash™ buffer (BD Biosciences) and stained with intracellular cytokines for an additional 30 min before washing and acquisition. Cytokine antibodies used were IL-4 FITC, IL-5 APC and IL-13 PE (all from BD Pharmingen. Flow cytometry was performed on a FACS Canto II flow cytometer with FACSDiva software v.6 (Becton Dickinson). The lymphocyte gating was set by forward and side scatter and 100,000 gated lymphocyte events were acquired. Data were collected and analyzed using Flow Jo software. All data are depicted as F_o of CD4⁺ T cells expressing cytokine(s). Frequencies following media stimulation are depicted as baseline F_o while F_o following stimulation with antigens or PMA/ionomycin are depicted as net F_o (with baseline F_o subtracted).