Anti il 33 antibody

Purified human CD34+ progenitor cells were derived from GCSF–treated peripheral blood cells of healthy donors. These cells were grown at 37°C with 5% CO2 in serum-free medium consisting of Iscove’s modified Dulbecco’s medium (IMDM) with 1-thioglycerol, BIT9500 supplement (BITS) (Stem Cell Technologies), BSA (Sigma-Aldrich), and the indicated cytokines (PeproTech). The cells initially underwent 72 h of expansion with 100 ng/ml SCF (PeproTech), 100 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3 ligand) (PeproTech), 100 ng/ml thrombopoietin (TPO) (PeproTech), and 50 ng/ml IL-3 (PeproTech). After expansion cells were then seeded in erythroid differentiation medium, which contains recombinant human erythropoietin at 4.5 U/ml (Procrit; Amgen), 10 ng/ml SCF and BIT9500 supplement. Human bronchial epithelial cells (Beas2B) were grown at 37°C with 5% CO2 in DMEM medium with 10% FBS. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies, reverse transcriptase, and real-time PCR SosoFast reagents were purchased from Bio-Rad Laboratories (Hercules, CA). Amicon Ultra centrifuge filters were purchased from EMD Millipore (Billerica, MA). Anti-IL-33 antibody was from R&D Systems (Minneapolis, MN). Anti-LarminA/C antibody was from Santa Cruz (Dallas, TX). Anti-GAPDH and β-actin antibodies and RBC lysis buffer were from Sigma (St. Louis, MO).