Chemotaxis Assays

Eosinophils, purified from BAL fluid of OVA-sensitized and -challenged wild-type or LTC₄S^−/− mice, were resuspended at a concentration of 1 × 10⁶ cells/ml in RPMI 1640 with 0.1% OVA, 24 mM HEPES, and 2 mM glutamine and pre-incubated for 30 minutes with LTC₄ (200 nM) or control medium. Transwell permeable supports containing polycarbonate membranes with 5 µm pores were used (Corning, Lowell, MA). The lower chambers below the transwell contained 600 µl of RPMI-1640 containing 0.2% bovine serum albumin with or without recombinant CCL19 or CCL21 (R&D Systems, Minneapolis, MN) or sphingosine-1-phosphate (Sigma). Eosinophil suspensions (100 µl) were added to the upper chamber. Wells were set up in triplicate for each experiment. Chemotaxis plates were incubated at 37°C for 2 hours. Migrating cells in the lower chamber were counted for 15 seconds in triplicate by flow cytometry.