Anti mouse il 17 pe

Cells were isolated according to an established method with slight modifications. Briefly, the spleen of the mice was harvested 28 days after the first immunization, pushed through a sieve, single cell suspension was prepared. Then, cells were isolated by density centrifugation on Histopaque 1077 (Sigma, St. Louis, MO, USA). Mononuclear cells were collected, washed with PBS containing 0.5% BSA, pre-incubated for 15 minutes with unlabeled isotype control Abs (IgG1 or IgG2b) (eBioscience, San Diego, CA, USA), and then surface-stained with anti-mouse CD3-FITC/CD4-PerCP or CD4-PerCP/CD25-PE-cy7. After the surface staining, cells were fixed, permeabilized and stained with anti-mouse IL-17-PE or Foxp3-PE (eBioscience, San Diego, CA, USA). Then, cells were incubated on ice for 30 minutes followed by washing with PBS. Subsequently, the cells (1 × 10⁶) were analyzed using the ASR α flow cytometry system (Becton Dickinson, Franklin Lakes, NJ).