Liver ROS Quantification with DCFDA

ROS level in the liver was determined using the fluoresenct probe dichlorofluorescein (DCF) as previously described [25 (link)]. The stock solution of 2’,7’-Dichlorofluorescin diacetate (DCFDA, D6883, Sigma-Aldrich, St. Louis, MO) was prepared by dissolving DCFDA in 12.5 mM of ethanol and stored at -80°C until used. Approximately 20 mg of liver was homogenized in 0.5 ml HEPES buffered saline (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose), centrifuged at 1000 × g for 10 min. Homogenate containing 100 μg protein was pipetted into a black 96-well plate. The DCFDA was diluted to 125 μM immediately before use and pipetted into each well to a final concentration of 25 μM. The plate was placed on a shaker for 2 min and incubated at 37°C in the dark for 30 min. The fluorescence was read on a Wallac Victor 3 Multilabel Counter (PerkinElmer Inc., Waltham, MA) at excitation 485 nm/emission 530 nm at 0 and 50 min. The ROS level was expressed as the increased absorbance value between 0 and 50 min. Mean of fluorescence intensity was calculated from five mice per group.