4.7. Measurement of Mitochondrial ROS Production Using MitoSOX Red

Mitochondrial superoxide production was measured using the MitoSOX Red fluorescent probe according to [137] with modifications. Cells were plated in 8 replicates into a black 96-well cell culture plate at a density of 1.5 × 10⁴ cells/well. After 24 h incubation in the presence of tested compounds, cells were washed twice with HBSS to remove the medium and subsequently incubated for 10 min (needed to allow the probe to enter the cell and start the reaction within the mitochondria) at 37 °C in 100 µL of measurement buffer containing 2.5 µM MitoSOX Red. After the incubation, the cells were washed twice with HBSS. The fluorescence was monitored in the measurement buffer with a Tecan Infinite M200 plate reader (Tecan US Inc., Durham, NC, USA) set to 510 nm excitation (Ex bandwidth: 10 nm) and 595 nm emission (Em bandwidth: 35 nm) wavelengths.