Collection and analysis of the BAL
Bronchoalveolar lavage was performed 24h after the last allergen challenge, by intratracheally injecting and aspirating 0.8 ml saline twice. After its collection the BALF was centrifuged for 5 min at 1500 rpm.
The cell pellets were resuspended in 1 ml PBS and an aliquot was stained with trypan blue solution and cells were counted using a Neubauer chamber. Eosinophils and neutrophils were detected by fluorescence-activated cell sorting (FACS) analysis. The cell surface staining was performed with antibodies against CD3 (eBioscience, Frankfurt, Germany), GR-1 (BD Bioscience, Heidelberg, Germany), CD45R (eBioscience, Frankfurt, Germany) and CCR3 (BD Bioscience, Heidelberg, Germany) for 30 min at 4°C.