Anti il 17 fitc

LPLs and pLN cells stimulated for 4 hours with PMA (50 ng/ml; Sigma), ionomycine (1 μg/ml; Sigma), and the Golgi-traffic inhibitor Brefeldin (1 μl/ml; BD Biosciences). Cells were stained with anti-CD3-PE (BD Pharmingen) or anti-CD3-APC (eBioscience) and anti-CD4-APC (Biolegend) or anti-CD4-FITC (BD Pharmingen). Next, the cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience). For intracellular staining the cells were incubated in permeabilization buffer (eBioscience) containing anti-IL-17-FITC (Biolegend), anti-IFNγ-FITC (BD Pharmingen), anti-IL-4-PE (BD Pharmingen) or Foxp3-FITC (eBioscience). An appropriate isotype matched control antibody was used in all FACS analyses. Cells were analyzed on a FACS Calibur using the CellQuest software (BD Biosciences). Results were analyzed with FlowJo version 7.6.5.

1.0 × 10⁶ PBMCs were stimulated with 1 μg/ml mesothelin peptide pool (Peptides & Elephants, Potsdam, Germany), medium control or PMA/Ionomycin (positive control, Sigma-Aldrich, St Louis, MI; USA) in R10 medium in the presence of Brefeldin A (10μg/mL, Sigma-Aldrich St Louis, MI, USA) for 6 hours at 37°C. Stimulation was stopped by transferring the cells to a 4°C refrigerator, followed by washing with FACS buffer and staining with the following reagents: anti-CD3 Pacific blue (BD Biosciences, CA, USA), anti-CD4 PerCP-Cy5.5 (BD Biosciences, CA, USA) and anti-CD8 APC-Cy7 (BD Biosciences, CA, USA). After a 15-minute incubation at 4°C, the cells were washed and fixed with a Fix/Perm reagent (Beckman coulter, CA, USA), followed by a further 30-minute incubation at 4°C with an intracellular antibody mix (anti-TNFα APC (BD Biosciences CA, USA), anti-IFN-γ PE-Cy7 (BD Biosciences CA, USA), anti-IL-2 PE (BD biosciences CA, USA) and anti-IL-17 FITC (BioLegend, CA, USA)). The stained cells were then washed with FACS buffer and acquired on a FACSAria flow cytometer (BD Biosciences, Stockholm, Sweden). Data was analysed using FlowJo software (Treestar Inc.).