Pe labeled anti il 17a

Mouse skin-draining lymph nodes (LNs; axillary and brachial lymph nodes) were harvested immediately after sacrifice and dissociated using a 70-um cell strainer (SPL Life Sciences, Pocheon, Korea). LN cells were cultured with 1X Protein Transport Inhibitor Cocktail (500×, eBioscience, San Diego, CA) and 1X Cell Stimulation Cocktail (500×, eBioscience) for 16 hours to stimulate and expand the T cells. Mouse interferon gamma (IFN-γ), IL-4 and IL-17A were subsequently assessed by flow cytometry^{62 (link)}. The LNs were then stained using FITC-labeled anti-IFN-γ, PERCP-CY5.5-labeled anti-CD4, APC-labeled anti-IL-4, PE-labeled anti-IL-17A and the respective isotype controls (eBioscience) to analyze CD4⁺ IFN-γ, IL-4 and IL-17A expression in accordance with the mouse Th1/Th2/Th17 Phenotyping Kit general protocol (BD Biosciences, San Jose, CA). The stained cells were then analyzed by FACS Canto with BD FACSDiva 8.0.1 (BD Biosciences, Mountain View, CA).

APC-Cy7-labeled anti-CD3, PE-Cy7-labeled anti-IL-10, and isotype-matched control Igs were obtained from Biolegend (San Diego, CA, United States). PE-labeled anti IL-17A, and isotype-matched Ig control were obtained from eBioscience [San Diego, CA, United States]. FITC-labeled anti-CD4, PE-CF594-labeled anti-CD8 and isotype-matched control Ig were obtained from Becton Dickinson Horizon (San Jose, CA, United States). PerCP-labeled anti-LAP [TGF-β1] and isotype-matched control Ig were obtained from R&D Systems (Minneapolis, MN, United States). APC-labeled anti-Foxp3, the Foxp3 staining buffer set, and isotype-matched control Ig were obtained from eBioscience (San Diego, CA, United States). LIVE/DEAD ® Fixable Aqua Dead Cell Stain Kit was obtained from Life Technologies (Carlsbad, CA, United States). Phorbol-12-myristateacetate (PMA) and ionomycin were obtained from Sigma-Aldrich. FITC-labeled anti-CD4, isotype-matched control Igs, and Monensin solution (Golgi Stop) were obtained from BD Pharmingen (San Jose, CA, United States). For confocal microscopy imaging, polyclonal rabbit anti-CD4 (Novus, Colorado, United States), Donkey anti-rabbit-AlexaFluor-568 (Abcam, Cambridge, United Kingdom), monoclonal mouse anti-human LAP (R&D Systems) and goat anti-mouse AlexaFluor-647 (Abcam) were used.