Bvd6 24g2

To determine the CD4⁺ T cell populations in the lung, single cells were isolated as above and stimulated for 5 h in RPMI-1640 (HyClone) containing 10% heat inactivated fetal bovine serum (FBS; Life Technologies), 100 U/ml penicillin (HyClone), 100 mg/ml streptomycin (HyClone), 5 ng/ml phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich), and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of a protein transport inhibitor (1 μl/106 cells; GolgiPlug, BD Biosciences, Franklin Lakes, NJ). After stimulation, the cells were stained with fixable viability dye, fixed and permeabilized, and labeled with antibodies to CD3 (17A2), CD4 (RM4-4; Biolegend), IFNγ (XMG1.2), IL-4 (BVD6-24G2) and IL-17 (17B7; eBioscience). Flow data were then acquired on a Canto II flow cytometer (BD Biosciences) and analyzed and plotted with FlowJo software (v10 for Windows; Tree Star; Ashland, OR, USA). All antibodies and viability dye were from eBioscience (San Diego, CA, USA) unless otherwise stated.
To determine the myeloid dendritic cell (mDCs) responses in the lung, single cells were isolated as aforementioned and stained by antibodies against CD11c (N418), MHCII (M5/114.15.2), CD80 (16-10A1), and CD86 (GL1). Flow data were then acquired, analyzed and plotted as above.

IL-4 levels in culture supernatants were determined by a sandwich ELISA specific for murine IL-4 using paired capture and detection antibodies (clone 11B11 and biotin-conjugated clone BVD6-24G2; eBioscience) and developed by streptavidin-HRP conjugate (R&D Systems, Minneapolis, MN) and ABTS substrate (Roche, Indianapolis, IN) as previously described [42 (link)]. Cytokine concentrations were calculated based on a standard curve generated using serial dilutions of murine recombinant IL-4 (R&D Systems). In some experiments, cytokine levels were determined in supernatants of AWH-stimulated MLN cells or serum using a custom-made cytokine Bio-Plex Pro 10-Plex assay (Bio-Rad, Hercules CA) according to the manufacturer’s instructions. Acquisition was performed on the MAGPIX platform (Luminex) and data were analyzed using the Bio-Plex Manager 6.1 software (Bio-Rad). To determine TGFβ levels, supernatants were acid-activated and neutralized prior to assay using an ELISA performed according to the manufacturer’s instructions (Mouse TGFβ DuoSet, R&D Systems).