*3.11. Eosinophil Preparation*

Enriched preparations of eosinophils were obtained from the peripheral blood of IL-5-transgenic mice. Eosinophil-enriched cells were obtained by the Percoll density gradient separation method described previously [19], with modification. Briefly, isotonic Percoll was prepared using a 10× solution of Krebs Ringer PBS (KRP; 10 mM sodium phosphate buffer, pH 7.5, containing 154 mM NaCl, 6 mM KCl and 1 mM MgCl2) and diluted with KRP to achieve concentrations of 60%, 70% and 80%. In 15- mL conical polypropylene tubes (BD Falcon 352096, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 2 mL of cell suspensions of 20 to 50 × 106 cells in KRP were placed, followed by the careful layering of aliquots (2.5 mL) of each concentration of Percoll solution starting with the lowest concentration at the bottom. The tubes were spun at 1000× *g* for 20 min at room temperature. Eosinophils were extracted from the 70% to 80% Percoll fractions by removing B and T lymphocytes using anti-B220- and anti-Thy1.2-coupled Dynabeads (DYNAL A.S., Oslo, Norway). Briefly, lymphocytes bound to these beads were removed using a permanent magnet. Unbound cells were used as an eosinophil-enriched fraction. To identify eosinophils, aliquots were removed and assessed using eosinophil  peroxidase staining as described previously [19]. More than 93% of the cells prepared by this method were eosinophils. 
