The samples were then incubated with 50 µl/well of OVA-biotin (1 mg/ml; Sigma, St Louis, MO, USA) at room temperature for 1 h. Plate-bound antibody was detected by treatment

with 50 µl/well of streptavidin–horseradish peroxidase (1 : 10 000; Southern Biotechnology) for 1 h at room temperature. The colour reaction was developed by adding 100 µl/well of 200 pmol of OPD (Sigma) in pH 5·0 citrate phosphate buffer plus 0·04% H2O2 for 10 min and stopped with 50 µl of 5% sulphuric acid per well. The plates were read at 492 nm in an ELISA reader (Bio-Rad, Hercules, CA, USA). The lungs of five mice per group were removed and treated with 100 U/ml of collagenase from Clostridium histolyticum (Sigma) for 30 min at 37°C. Subsequently, the digested lung tissue was filtered through a 70 micrometre cell strainer and the red blood cells were lysed with ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2; Invitrogen, CA,

USA). The cell selleckchem suspension was washed twice in RPMI-1640 and adjusted to 1 × 106 cells per well for surface staining and to 2·5 × 106 cells for the intracellular cytokine experiment. For CD4 and forkhead box P3 (FoxP3) staining, the cells were generally blocked with anti-mouse CD16/CD32 monoclonal antibodies (mAbs) (Fc-block) and stained for surface marker using fluorescein isothiocyanate (FITC)-labelled anti-mouse CD4 (BD Bioscience) mAb or isotype control, which were incubated for 20 min at 4°C with antibody dilution Y 27632 solution (PBS 0·15 M, 0·5% BSA, 2 mM NaN3). The cells were then washed with 0·15 M PBS and incubated with strepatavidin–phycoerythrin–cyanine 5 (PE-Cy5) (1 : 200) Aspartate for an additional 20 min at 4°C. Surface-stained cells were washed twice with 0·15 M PBS and incubated with fixation/permeabilization buffer (eBioscience) for 30 min at

4°C. Anti-FoxP3-PE-labelled antibodies in permeabilization buffer (eBioscience) were added to cells and then incubated for 30 min at 4°C. Cells were washed twice with 150 µl of permeabilization buffer (eBioscience) and fixed with 2% paraformaldehyde. For IL-10 and FoxP3 intracellular staining, cells were cultured for 14 h in medium or OVA (25 µg/ml). After this stimulation period, 1 mg/ml of brefeldin A was added to the cell culture, which was incubated for an additional 4 h in a CO2 incubator at 37°C. Before CD4 staining, the cells were treated with anti-CD16/CD32 (Fc-block). Cell surface and intracellular staining were performed as described above for surface experiments; however, the cells were stained for CD4, IL-10 and FoxP3 using anti-CD4 FITC-labelled, anti-IL-10 PE-labelled, and anti-FoxP3 biotin-labelled plus streptavidin–PE-Cy5 antibodies. Data acquisition was performed using fluorescence activated cell sorter (FACScan) (Becton Dickinson, San Jose, CA, USA). Data analysis was performed using a FlowJO interface (Becton Dickinson). Statistical analysis was performed using the software GraphPad Prism (GraphPad Software, San Diego, CA, USA). The mean ± standard deviation (s.d.