Quantifying Mitochondrial Content in Liver

MTDR mitochondrial content quantification was performed as published [19 (link)]. Briefly, 20 μl of liver homogenate (8 μg) were seeded per well on a clear-bottom black 96-well plate (Corning) in 100 μl of MAS (see 2.5) containing MitoTracker Deep Red (MTDR, Thermo Fisher) at 0.5 μM final concentration. This liver homogenate with MTDR was incubated at 37 °C for 10 min. Plates were centrifuged at 2,000 g for 5 min at 4 °C (no brakes), and supernatant was carefully removed. Finally, 100 μl of MAS was added per well. MTDR was excited at 625 nm and its emission recorded at 670 nm. Mitochondrial content was calculated as MTDR signal, minus background fluorescence (from wells without protein loaded), per milligram of protein. Fluorescence was measured using the Spark 20 M Microplate Reader (Tecan).