Biotek synergy neo microplate reader

The generation of mitochondrial superoxide was measured with MitoSOX Red (Invitrogen, Life Technologies, Carlsbad, CA, USA). First, the cells were incubated with MitoSOX Red at a concentration of 5 μM for 10 mins at 37°C and washed three times with Hanks Balanced Salt Solution (HBSS). The images were captured using a Bx51 fluorescence microscope (Olympus, Tokyo, Japan), and the level of fluorescence was detected using BioTek Synergy NEO microplate reader (BioTek, Vermont, USA) at 510/580 nm. For in vivo experiments, freshly isolated, frozen, and non-fixed mouse livers were incubated with MitoSOX Red (5 μM) for 10 mins at 37°C after being sectioned by cryostat at 10 μm. Then, the solution on the slides was removed, and slides were imaged with an inverted confocal microscope FV1200 (Olympus). Finally, the level of fluorescence was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Tetramethylrhodamine, methyl ester (TMRM) (Invitrogen) was used to measure the mitochondrial transmembrane potential. Notably, the signal is bright in healthy cells with functioning mitochondria. Hoechst 33342 was used to stain nuclei in live L-02 cells. Cells were incubated with 100 nM TMRM for 30 mins at 37°C, after washing 3 times with HBSS, cells were then incubated with Hoechst 33342 for 10 mins at 37°C. The cells were incubated in DMEM until use and images were collected on an inverted confocal FV1200 microscope (Olympus). Finally, the fluorescence intensity was detected using BioTek Synergy NEO microplate reader (BioTek) at 548/574nm.