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

Assessing the physiological role of H(2)O(2) requires sensitive techniques to quantify H(2)O(2) 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 H(2)O(2), 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)