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After 4?h of incubation (37��C, 5% CO2), the cells in the lower chamber were counted using flow cytometry (FACScalibur; Beckton Dickinson, Cowley, Oxfordshire, UK). Relative cell counts were obtained by acquiring events for 60?s. Healthy non asthmatic subjects (n?=?25) and mild asthmatic subjects (n?=?15) were recruited from a research database of volunteers. flupentixol Ethical approval was obtained from the Southampton and South West Hampshire joint local research ethics committee and the volunteers gave their written informed consent. Detailed clinical history and physical examination were performed on all subjects. Spirometric tests and assessment of asthma severity were in accordance with the BTS/SIGN guidelines. All subjects were tested for atopy by skin prick testing and were free from respiratory tract infections for a minimum of 4?weeks before inclusion in the study. Subjects underwent fibreoptic bronchoscopy under local anesthesia as previously described (19) using find more an Olympus BFIT20 bronchoscope (Olympus, Tokyo, Japan). Bronchoalveolar lavage fluid was obtained and processed for cell count and cytokine measurement as previously described (19). Bronchoalveolar lavage fluid was concentrated 10�C30 fold using Amicon Centripep YM-3 centrifugal filter devices (Millipore Ltd, Watford, UK) before quantification of OPN. Levels of OPN in eosinophil supernatants and in BAL fluids were measured using an ELISA kit (Quantikine; R&D, Systems, Minneapolis, MN, USA). The lower limit of assay sensitivity was determined according to the manufacturer��s instructions. Data are expressed as Mean?��?SEM of at least three independent experiments. Parametric anova, followed by Tukey�CKramer multiple comparisons test, was used to compare groups. Pearson and Spearman correlation tests were used for correlating normal and nonparametric variables, respectively. Mann�CWhitney selleck chemical Rank sum test was used as statistical method to compare OPN in BAL from normal and asthmatic subjects. A probability of