Anti il 17 pe cy7

Cells were cultured in 96-well flat-bottom plates in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (all from Thermo Fisher Scientific). Cells were stimulated with PMA (50 ng/mL, Sigma-Aldrich) and ionomycin (1 μg/mL, Sigma-Aldrich) for 4 h. GolgiPlug (0.67 μg/mL, BD Biosciences) was added during the final 2 h. After the cell-surface antigens were stained, intracellular cytokines were stained using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences), anti-TNF-PE-Cy7, anti-IFNγ-PE-Cy7, anti-IL-17-PE-Cy7, and anti-CD40L-PE-Cy7 mAb (all from BioLegend) or their isotype control antibodies.

As previously described [35 (link)], single-cell suspensions of corneal samples were prepared by collagenase digestion and blocked with anti-FcR mAb for 30 minutes at 4°C in 1% BSA in PBS. The isolated cells were stained with the following antibodies: anti-CD11b APC, anti-CD45 PE, anti-CD19 PE/cy7, and anti-CD4 FITC (Biolegend). All antibodies were analyzed with appropriate isotype controls. Cells were analyzed using FACSCanto and FACSAria flow cytometers (BD Biosciences, San Jose, CA, USA). Additional cornea and conjunctiva samples were prepared for intracellular cytokine staining with anti-CD207 APC, anti-IFN-γ FITC, and anti-IL-17 PE/cy7 (BioLegend) according to the manufacturer’s instructions.

Cells were incubated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma), ionomycin (1 μg/ml; Sigma), and Brefeldin A (1 μl/ml; BD Biosciences) at 37 °C for 4 hours and stained with anti-CD4-APC (Biolegend), anti-TCRβ-FITC (Biolegend) and fixable viability dye (eBioscience). For intracellular staining, cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience) and stained with anti-IL-17-PECy7 (biolegend), anti-IFNγ-PE (BD pharmingen), and anti-FOXP3-PE (eBioscience) in permeabilization buffer (eBioscience). Data were collected on Gallios flow cytometer (Beckman Coulter) and analyzed with Flowjo 10.0 software.