The thermal cycling conditions were: 30 sec at 95°C for initial denaturation, followed by 40 cycles of 5 sec at 95°C, 30 sec at 60°C for amplification, and 15 sec at 95°C, 1 min at 60°C and 15 sec at 95°C for melting curve analysis. Target gene primers are presented in Additional file 8: Table S3, in the supplemental material. An untreated cell sample was used as the calibrator and the fold-change for this sample was set as 1. Target gene Ct values were normalized to β-actin, and the results were analyzed by means of the 2-△△Ct method [60]. Measurement of IL-33 cytokine by enzyme linked immunosorbent assay Peripheral blood and

bronchoalveolar lavage fluid (BALF) samples of 30 pediatric patients with MPP (aged from 2.08-8.75 years old) were collected from Children’s Hospital, Zhejiang University School of Medicine from January 2012 to December 2012. Samples Selleckchem Ipilimumab from age-matched children (aged from 2.50-8.50 years old) with foreign body in bronchus were used as controls. All samples were collected with informed consent from their guardians. This study was approved by the Ethics Committee of the Children’s Hospital, Zhejiang University School of Medicine. AZD1208 mw The procedure of fiberoptic bronchoscopy (FOB) and BALF collection were performed as described previously [61]. The samples were centrifuged at

2000 g for 10 min, and the supernatants were stored at -80°C until analysis. The levels of IL-33 in serum and BALF were determined using the IL-33 enzyme-linked immunosorbent ID-8 assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. Statistical analysis Each experiment was repeated at least three times independently. Data were expressed as mean ± SD and

evaluated with Student’s t-test or Mann–Whitney U test. p < 0.05 was considered statistically significant. Acknowledgments Jun Yang is a recipient of the Zhejiang Provincial for the Cultivation of High-level Innovative Health Talents. The work was supported by grants from the National Nature Science Foundation of China (Nos. 81070004, 81000765, 81172692, 81373036); and Zhejiang Provincial Natural Science Foundation (No. LY12H2600). The authors have no conflict of interest to declare. Electronic supplementary material Additional file 1: Figure S1: Assessment of A549 cell growth in serum-free medium. (A) Relative viability of cells was determined by the MTT assay. Mean absorption was normalized to control, with controls (untreated + SM group) being 100%. (B) Cell growth rate was investigated by cell count. (C) Cell viability was measured by Trypan blue exclusion assay. (D) Micrographs (200×) of cell morphology. The values represent averages of three independent experiments with six replicate detections (mean ± SD). *, M.