Quantifying mitochondrial redox and bilirubin content in intact primary hepatocytes of obese mice using fluorescent reporters

Abstract

Summary: Assessing the physiological role of H2O2 requires sensitive techniques to quantify H2O2 and antioxidants in live cells. Here, we present a protocol to assess the mitochondrial redox state and unconjugated bilirubin levels in intact live primary hepatocytes from obese mice. We described steps to quantify H2O2, GSSG/GSH, and bilirubin content in the mitochondrial matrix and the cytosol using the fluorescent reporters roGFP2-ORP1, GRX1-roGFP2, and UnaG, respectively. We detail hepatocyte isolation, plating, and transduction and live-cell imaging using a high-content imaging reader.For complete details on the use and execution of this protocol, please refer to Shum et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Metabolism
• Molecular Biology