Il 23r

Freshly isolated ear tissue was diced and subsequently digested with 0.25 mg/ml Liberase TL (Roche) at 37 °C for 135 minutes. Epithelial fragments were removed by filtration (EASYstrainer 70 μm mesh, Greiner Bio-one, Kremsmünster, Austria). Lymph nodes were passed over a 40 μm cell strainer (EASYstrainer, Greiner Bio-one) and washed with PBS. Single-cell suspensions were stained with Live/Dead Fixable Aqua dead cell stain kit (Invitrogen, Waltham, MA) for 20 minutes to exclude dead cells and treated with Fc-blocking anti-mouse CD16/32 antibodies (TruStain FcX; BioLegend). Cells were subsequently stained with fluorochrome-conjugated antibodies against the following surface antigens: CD45, CD3, NK1.1, CD49a, CD49b, CD44, CCR6, CD27, CD11b, CX3CR1, CXCR3, CD127, CD103, IL-23R (all BioLegend), and IL-12Rβ2 (R&D Systems, Minneapolis, MN).

After staining with live/dead dye for 15 minutes at 4°C, surface markers (Biolegend, San Diego, CA, USA) were used to label the harvested cells: CD4 (catalog 100434), CD25 (catalog 102016), IL-23R (catalog 150907), CXCR3 (catalog 126506), and CCR6 (catalog 129819). For intracellular labeling, cells were cultured for 4 hours with PMA (5 ng/mL), Brefeldin A (1000 ng/mL), and ionomycin (500 ng/mL), followed by fixation and permeabilization before labeling with IL-22 (catalog 516409), IL-17A (catalog 506930), IFN-γ (catalog 505808), Foxp3 (catalog 11-5773-82), granulocyte-macrophage colony-stimulating factor (GM-CSF; catalog 2131415), and IL-10 antibodies (catalog 505007). Finally, the cells were measured by flow cytometry and further analyzed using FlowJo software (version 10.0.7).