Anti il17 clone ebio17b7

Single-cell suspensions were prepared from inguinal lymph node extracts. Surface antigen staining was performed using the following antibodies (eBioscience): anti-CD45 (clone 30-F11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53–6.7), and anti-TCRγ (clone H57-597). For intracellular cytokine staining, isolated cells were activated in complete RPMI containing 0.5 µg ml⁻¹ phorbol 12 myristate 13-acetate (PMA), 0.5 µg ml^-1 ionomycin and 10 µg ml⁻¹ brefeldin A (all from Sigma-Aldrich) for 4 h. After surface staining, cells were fixed and permeabilized using Cytofix/Cytoperm solution (BD). Cells were washed in perm/wash buffer and incubated for 30 min at 4°C with different antibody combinations specific for murine cytokines (all from eBioscience) diluted in perm/wash buffer: anti-Il4 (clone 11B11), anti–IL-13 (clone eBio13A), anti-Il17 (clone eBio17B7), and TNF (clone MP6-XT22). Immunostained cells were analyzed by flow cytometry using a FACS Aria flow cytometer (BD).