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.