1b). The lungs were washed by cannulating the AZD4547 research buy trachea and gently injecting/recovering (3×) 1·0 ml of PBS. The bronchoalveolar lavage fluid (BAL) was centrifuged at 300 g at 4°C for 5 min and the supernatants were stored at −20°C for cytokine analysis. The cell pellet was resuspended in 0·1 ml of 3% bovine serum albumin (BSA) and cells counted using a haemocytometer. The cells were then cytocentrifuged and stained with haematoxylin and eosin (H&E) for differential

counting based on cell morphology and staining patterns. The means of three independent counts of 100 cells in a randomized field were shown. Following bronchoalveolar lavage, the lungs were fixed with formalin. Serial sagittal sections of whole lung (3–4 µm Nutlin-3 mw thick) were cut and stained with Gomori trichome for light microscopy. At least 10 fields were selected randomly and examined. The severity of the inflammatory process in the lungs was scored by two pathologists who were blinded to group identity. The scale varied from 0 to 5 as follows: 0, no inflammation, 1, minimal; 2, mild; 3,

medium; 4, moderate; and 5, marked [35,36]. The EPO assay was performed as described previously [37]. Briefly, a 100-mg sample of tissue from each lung was homogenized in 1·9 ml of PBS and centrifuged at 12 000 g for 10 min. The supernatant was discarded and the erythrocytes were lysed. The samples were centrifuged, the supernatant discarded and the pellet resuspended in 1·9 ml of 0·5% hexadecyltrimethyl ammonium bromide in PBS saline. The samples were frozen in liquid nitrogen and centrifuged at 4°C at 12 000 g for 10 min. The supernatant was used for the enzymatic assay. Briefly, o-phenylenediamine (OPD) (10 mg) Suplatast tosilate was dissolved in 5·5 ml distilled water, and then 1·5 ml of OPD solution was added to 8·5 ml of Tris buffer (pH 8·0), followed by addition of 7·5 µl H2O2. In a 96-well plate, 100 µl of substrate solution was added to 50 µl of each sample. After 30 min, the reaction was stopped with 50 µl of 1 M H2SO4 and the absorbance was read at 492 nm. Levels of IL-4, IL-5,

IL-10, TNF-α and IFN-γ were determined by bronchoalveolar lavage (BAL) of the different groups of mice with an enzyme-linked immunosorbent assay (ELISA) sandwich technique using commercially available kits (OptEIA; BD Bioscience, San Jose, CA, USA), according to the manufacturer’s protocol. The optical density (OD) values were read at 450 nm. The results were expressed as picograms per millilitre, compared to a standard curve. The levels of OVA-specific IgE in serum were determined by ELISA, as described previously [38,39]. Briefly, Maxisorp 96-well microtitre plates (nunc, Roskilde, Denmark) were coated with rat anti-mouse unlabelled IgE (1 : 250; Southern Biotechnology, AL, USA) in pH 9·6 carbonate-bicarbonate buffer for 12–16 h at 4°C and then blocked for 1 h at room temperature with 200 µl/well of 0·25% PBS-casein.