Hard Info About CHIR-99021 Unveiled
Cells were analyzed for their fluorescence intensity using FACS Calibur (Beckton Dickinson). Results are expressed as mean fluorescence intensity (MFI) or percentage of positive cells. BSM cells were stimulated with optimal concentrations of IL-1��, TNF-��, IL-6 (all from R&D Systems), or SLIGKV-NH2 (Sigma-Aldrich, Saint Quentin-Fallavier, France), the Fleroxacin agonist peptide of protease activated receptor 2 (PAR-2), for 72?h. Mast cells were challenged with optimal concentrations of TGF-��1 (transforming growth factor-beta 1), SCF (stem cell factor), RANTES (regulated upon activation normal T cell expressed and secreted), fractalkine (all from R&D Systems), PMA (phorbol 12-myristate 13-acetate) or calcium ionophore A23187 (both from Sigma-Aldrich) for 1?h at 37��C. HMC-1 or HLMC adhesion to BSM cells was initially assessed using radiolabeled mast cells as adapted from Lazaar et?al. (21) using pulsed mast cells with 10?��Ci/ml [3H]-thymidine (Amersham Biosciences, Orsay, France). In another set of experiments, mast cells were allowed to adhere to a monolayer of BSM cells for 1?h at 37��C in 8-well chamber slides or cell culture inserts (VWR international, Strasbourg, France). Mast cells and BSM cells were stained respectively with Texas Red?-conjugated anti-tryptase (Dako) and FITC-conjugated anti-��-smooth muscle actin Abs (Molecular Probes, Eugene, OR, USA). Confocal images were obtained using Fluoview laser scanning microscope (Nikon, Paris, France) and reconstituted in 3D images using Imaris Software (Bitplane, Z��rich, Switzerland) as described previously (6). Electronic images from inserts Y27632 were obtained using Philips 301 electron microscope (Philips Electron Optics, Eindhoven, The Netherlands) (7). Total RNA was extracted from BSM cells and mast cells as described previously (31). After reverse transcription, real-time quantitative PCR was performed on a Rotor-GeneTM 2000 (Corbett Research, Mortlake, Sydney, Australia), using appropriate primers (Sigma-Aldrich) designed to target CD51, CD44 or collagen type I ��1. Results were normalized by the geometric mean of three internal housekeeping genes (32). Western see more blot was performed on cell protein extracts (26) using primary antibody directed against collagen type I �� monoclonal Ab (Santa Cruz, Heidelberg, Germany). Blot images were acquired using biocaptMW software (Thermo Fisher Scientific). Band densities were quantified using ImageJ software and normalized to that of ?-actin. Values are presented as the mean?��?the SEM. Statistical analysis was performed using ncss 2001 software. Statistical significance was analyzed by anova, Kruskal�CWallis and paired Student��s t-test. P-value
