Ethanol exposure promotes the development of steatohepatitis which can progress to

Ethanol exposure promotes the development of steatohepatitis which can progress to end stage liver disease. sirtuin-3 activity thereby preventing LPS and ethanol from stimulating the binding of hexokinase II to the mitochondria and precluding NADPH oxidase and inflammasome activation. (20). Livers were perfused with 0.05% collagenase and the resulting suspension of liver cells was treated with 0.02% Pronase for 15 min at 12 °C. The resulting cell suspension from two rats per treatment group was pooled and then centrifuged three times at 100 × for 2 min. The pooled supernatant was then purified by centrifugal elutriation. The Kupffer cells were suspended in CMRL medium. After 1 h non-adherent cells were removed by aspiration and fresh medium was added. Measurement of IL-1β and TNFα Cell culture medium was removed at the times indicated and stored at ?20 °C for TNF-α or IL-1β assay using ELISA (R&D Systems Minneapolis MN). High binding capacity polystyrene 96-well plates were coated with purified biotin-conjugated anti-murine IL-1β or TNF-α antibody (1 μg/ml) overnight. Avidin-HRP was then added at 1:5 0 for 30 min at room temperature followed by 100 μl/well 3 3 5 5 substrate. values were read at 450 nm with a 570-nm subtracted correction using a BioTek? plate reader. Measurement of Caspase-1 Activity The activity of caspase-1 was measured in cell lysates using the fluorometric substrate Ac-YVAD-AFC. Kupffer cells were washed with ice-cold phosphate-buffered saline Catechin (PBS) and lysed in lysis buffer (50 mm Tris-HCl pH 7.4 150 mm NaCl 20 mm EDTA 0.3% Nonidet Catechin P-40 0.1 mm Na3VO4 1 mm PMSF 10 μg/ml leupeptin and 10 μg/ml aprotinin). Lysates were then centrifuged at 14 0 × for 10 min. The supernatants were collected mixed with 50 μl of reaction buffer (50 mm HEPES pH 7.4 100 mm NaCl 1 mm EDTA 10 sucrose 10 mm DTT and 100 μm Ac-YVAD-AFC) and then incubated at 37 °C for 1 h. Samples were read at 405 nm in a 96-well microtiter plate. Measurement of Reactive Oxygen Species Kupffer cells Catechin were cultured for 16-18 h and then stimulated with LPS at the times indicated at 37 °C in a 5% CO2 atmosphere. Mouse monoclonal to CD8/CD45RA (FITC/PE). Medium was then replaced with 100 μl of 5-(and-6)-carboxy-2′ 7 diacetate diluted in CMRL medium and 10% FBS and cells were incubated for 5 min in the dark. Fluorescence was measured using Catechin an excitation wavelength of 505 nm and emission detection wavelength of 530 nm. Translocation of p67phox to the Membrane Cells were washed with cold PBS with 1 mm sodium orthovanadate and homogenized in 20 mm Tris-HCl (pH 7.4) 1 mm EDTA and 250 mm sucrose with protease inhibitor mixture in a glass-on-glass Dounce homogenizer and centrifuged at 1 500 × for 15 min. The resulting supernatant was then centrifuged at 15 0 × for 15 min at 4 °C. The resulting supernatant was added to the PBL-specific ligand that binds to a specific plasma membrane protein (Qiagen Qproteome plasma membrane isolation kit). The resulting plasma membrane-enriched vesicles were precipitated using magnetic beads that bind to the PBL ligand. The plasma membrane vesicles were eluted under native conditions in buffer (50 mm Tris pH 7.4 1 Nonidet P-40 150 mm NaCl and 1 mm EDTA with protease inhibitor mixture). Samples were separated by SDS-PAGE and Catechin probed by Western blotting with antibody specific for p67phox. Western blots were probed with antibody to Na K-ATPase to ensure equal loading of plasma membrane proteins between samples. Mitochondrial and Cytosolic Isolation Kupffer cells from two individual wells (~1.0 × 106 cells total) were harvested and centrifuged at 600 × for 10 min at 4 °C. The cell pellets were resuspended in 3 volumes of isolation buffer (20 mm HEPES pH 7.4 10 mm KCl 1.5 mm MgCl2 1 mm sodium EDTA 1 mm dithiothreitol 10 mm phenylmethylsulfonyl fluoride 10 μm leupeptin and 10 μm aprotinin) in 250 mm sucrose and disrupted by 40 strokes of a glass homogenizer. The homogenate was centrifuged twice at 1 500 × at 4 °C to remove Catechin unbroken cells and nuclei. The mitochondrially enriched fraction (heavy membrane fraction) was then pelleted by centrifugation at 12 0 × for 30 min. The supernatant was removed and filtered through 0. 2-μm and then 0.1-μm Ultrafree MC filters.