HO-1 may facilitate the repair of injured tissues through inhibition of infiltrating inflammatory cells

-conjugated donkey anti-rabbit Adenovirus Transfection Cells were grown to 90% confluence, infected with Adeno-GFP and Adeno-C/EBPc, respectively, at 20 MOI. 24 h later, proteins were harvested for the analysis of C/EBPc expression. In some experiments, the cells were treated with 20 ng/ml IL-1b for the C/EBPc Suppresses IL-6 Production IgG. The membrane was developed by enhanced chemiluminescence technique according to the get 212141-51-0 manufacturer’s protocol. Isolation of Alveolar Type II Epithelial Cells Alveolar type II epithelial cells from C57BL6 mice were isolated and cultured using a method described previously. In all experiments, specific pathogen-free male C57BL/6 mice obtained from Jackson Laboratory were used. All procedures involving mice were approved by the Animal Care and Use Committee of Harvard Medical School. Using biotinylated CD32 and CD45 mAbs, lymphocytes were removed from alveolar type II epithelial cells by streptavidin-magnesphere. The methods achieved high yields, high purity and viability. Alveolar type II epithelial cells were identified by following methods: a) alkaline phosphatase staining, b) lamellar body identified by tannic acid staining and by transmission electron microscopy, c) immunochemistry for alveolar type II epithelial cells using monoclonal anti-pro-SP-C. Cells were also identified by pan-cytokeratin antibodies . We also used an improved method to maintain the primary cell culture with some modifications. Transmission Electron Microscopy TEM was used to characterize freshly isolated alveolar epithelial type II cells using modified Karnovsky’s fixative. Images were taken and analyzed according to our previous published methods. Immunostaining For immunocytochemistry, the cells were fixed in 3.7% paraformaldehyde and non-specific binding was blocked with blocking buffer for 30 minutes. Cells were incubated with primary antibodies at 1/300 dilution in blocking buffer for 1 h and washed three times with wash buffer. After incubation with appropriate fluorophore-conjugated secondary antibodies, the coverslips were mounted on slides with Vectashield mounting medium. The samples were observed on an LSM 510 Meta confocal microscope, and data were processed using the software provided by the manufacturer or Image J software. HEPES, 0.2 mM EDTA, 25% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and complete protease inhibitors. Protein concentrations were determined by Bio-Rad protein assay kit. The EMSA probes were double-stranded oligonucleotides containing a murine IL-6 C/ EBP binding site, or a NF-kB consensus oligonucleotide. C/EBP probes were labeled with a ATP. NF-kB probes were labeled with c ATP. DNA binding reactions were performed at room temperature in a 25 ml reaction mixture containing 6 ml of nuclear extract and 5 ml of 56 binding buffer Ficoll, 50 mM HEPES pH 7.9, 5 mM EDTA, 5 mM dithiothreitol). The remainder of the reaction mixture contained KCl at a final concentration of 50 mM, Nonidet P-40 at a final concentration of 0.1%, 1 mg of poly, 200 pg of probe, bromphenol blue at a final concentration of 0.06%, and water to final volume of 25 ml. Samples were electrophoresed through 5.5% polyacrylamide gels in 16 TBE at 190 V for approximately 3.5 h, dried under vacuum, and exposed to X-ray film. For supershifts, nuclear extracts were preincubated with antibodies for 0.5 h at 4uC prior to the binding reaction. The following antibodies were purchased from Santa Cruz, CA: NF-kB p5