Fluorometric sirt3 activity assay kit

To characterize the functional relevance of differences in myocardial SIRT3 abundance identified by proteomic analysis, we conducted additional in vitro SIRT3 gain and loss-of-function studies in adult primary ventricular cardiomyocytes (APVC) isolated from rat and AGS,^{16 (link)} respectively, and assessed the effects on cell viability following simulated I/R. Rat APVCs were treated with increasing concentrations of resveratrol (Sigma-Aldrich, St. Louis, MO), an activator of SIRT3, or nicotinamide, an inhibitor of SIRT3 (control, 5μM, 10 μM) for 1h. AGS APVCs were transfected with SIRT3-siRNA (60pmol) or scrambled RNA control (Santa Cruz, Dallas, TX) overnight, according to manufacturer's protocols. Cells were subjected to 2h of oxygen and glucose deprivation followed by 24h of reoxygenation and determination of apoptosis (Cell Death Detection ELISA kit, Roche, Indianapolis, IN) and necrosis (cardiac Troponin I levels in supernatant, Life Diagnostics, West Chester, PA). SIRT3 expression (Western blot) and activity (fluorometric Sirt3 activity assay kit, Abcam, Cambridge, MA) were measured in APVC lysates. All experiments were run in triplicate.