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发布于:2022-11-30 00:33:22  访问:7 次 回复:0 篇
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Ncubated for two hours at 37 in DMEM containing 2 FA-free BSA with or
In short, WAT was removed from Title Loaded From File anaesthetized WT and Ad-KO mice and homogenized in five volumes of chilled STE buffer (pH 7.four, 0.25 M sucrose, 10 mM Tris-HCl, and 1 mM EDTA). Immediately after this incubation period, this resuspended pellet ontaining reaction mixture was transferred to an Eppendorf tube, the cap of which housed a Title Loaded From File Whatman filter paper disc that had been presoaked with 1 mol/l sodium hydroxide. 14CO2 trapped within the reaction mixture media was then released by acidification of media working with 1 mol/l perchloric acid and gently agitating the tubes at 37 for 1 hour. Radioactivity that had turn into adsorbed onto the filter disc was then quantified by Title Loaded From File liquid scintillation counting in a -counter. FA synthase (FASN) activity assay. FASN activity was determined in tissues as previously described with slight modifications (60). Briefly, WAT from Ad-KO and WT mice was homogenized in tissue homogenization buffer (0.1 M Tris, 0.1 M KCl, 350 mM EDTA, and 1 M sucrose; pH 7.five) containing protease inhibitor cocktail (Roche). Tissue extracts were clarified by centrifuging at 9,400 g for 10 minutes at four . For figuring out FASN activity, BAT homogenate was added to NADPH activity buffer (one hundred mM potassium phosphate buffer, pH 7.five containing 1 mM DTT, 25 M acetyl-CoA, and 150 M NADPH). Malonyl-CoA (50 M) was added towards the reaction buffer to initiate the reaction. Raise in the absorbance was followed at 340 nm for ten minutes working with a spectrophotometer set in kinetic mode below continuous temperature (37 ). Activity of FASN is represented as nmoles NADPH consumed per minute per mg. Adipocyte size. For adipocyte size quantification and CD68 staining, Title Loaded From File perigonadal AT was fixed in zincformalin, mounted in paraffin blocks, reduce into 5-m sections, and stained with H E. The area of every adipocyte (in m2) was measured from tracing of all adipocytes inside a field of view with ImageJ software program from NIH (http://rsbweb.nih.gov/ij/). The typical adipocyte region for each and every group was derived from measurements of no less than one hundred adipocytes.Ncubated for 2 hours at 37 in DMEM containing two FA-free BSA with or without ten M isoproterenol. Medium samples had been collected to assay for FA working with a Wako NEFA-C-kit based on the manufacturer‘s protocol. The degree of FA was normalized towards the weight of adipose explants. In vivo lipolysis. In vivo lipolysis assays have been performed as previously described (58). Briefly, 7-hourfasted mice have been injected i.p. with saline and 15 minutes later blood was collected from the orbital plexus. Just after 2 days, the 7-hour-fasted mice had been injected i.p. with isoproterenol (ten mg/kg BW) and blood was collected following 15 minutes. Plasma nonesterified FA (NEFA) concentrations have been measured applying a Wako NEFA-C-kit based on the manufacturer‘s protocol. FA oxidation. FA oxidation was assayed as previously described (59). In short, WAT was removed from anaesthetized WT and Ad-KO mice and homogenized in five volumes of chilled STE buffer (pH 7.4, 0.25 M sucrose, 10 mM Tris-HCl, and 1 mM EDTA).
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