4.6. ROS Assay

The ROS assay was carried out using mitochondria isolated from liver and brain with the reactive oxygen species assay kit (MyBioSource, San Diego, CA, USA, MBS2540517) according to the manufacturer’s protocol manual. DCFH-DA (2,7-Dichlorofluorescein diacetate) is a fluorescent probe without fluorescence that can freely cross the cell membrane and can be hydrolyzed by intracellular esterase to form DCFH (dichlorofluorescin). In the presence of ROS, DCFH is oxidized to DCF (dichlorofluorescein) which is a strong green fluorescent substance that cannot penetrate the cell membrane. In short, 20 uM of DCFH-DA working solution was added to the mitochondrial pellet and incubated for 1 h at 37 °C. The mitochondrial suspension was then centrifuged, and the pellets were washed two times with the working reagent from the kit, then resuspended in the working reagent with equal volume and equal protein concentration on a microplate for fluorescence detection. Fluorescence at 500 nm excitation and 525 nm emission was then read on a fluorescence microplate reader (Synergy H1, BioTek, Winooski, VT, USA).