Chemotaxis assay

Blood was obtained from either sham- or Ang II–infused mice and total peripheral blood mononuclear cells were isolated by a standard density gradient with LSM 1077 Lymphocyte Separation Medium (PAA Laboratories, Pasching, Austria). Either T cells or B cells were isolated from peripheral blood mononuclear cells by negative selection. Cell purity was confirmed to be ≥96%. After separation, the lymphocytes were resuspended in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM/ml l-glutamine, and 50 μg/ml gentamicin (Sigma-Aldrich, St. Louis, MO, USA). Cells (2 × 10⁵) were added to the upper chamber of a 24-transwell apparatus (6.5 mm diameter, 5 μm pore size; Costar 3421) and incubated for 2 h at 37°C in 5% CO₂. Recombinant mouse CCL5/RANTES (10 ng/ml; R&D Systems, Minneapolis, MN, USA) or supernatant (conditioned medium) from an 18 h organ culture of pVAT (diluted 1:50) was placed in the lower chamber. The optimal concentration of RANTES was determined in preliminary experiments. In a subset of experiments, conditioned medium from pVAT–Ang II cultures was preincubated with 0.5 μg/ml anti-RANTES antibody (clone 53405; R&D Systems) chemotaxis was assessed. The percentage of cells that migrated to the lower chamber was determined by flow cytometry as described above.