Cells were treated with 5 μM mitoSOX for 10 min at 37 °C and then washed with PBS three times before visualizing with a Zeiss LSM 880 META microscope (Carl Zeiss MicroImaging, Inc.) with 561 nm excitation laser. Image analysis was conducted using Image J software.
Cells infected with retroviral vector pLPCX mt-roGFP2-Orp1 were visualized with a Zeiss LSM710 META confocal microscope (Carl Zeiss MicroImaging, Inc.) with excitation 405 at 488 nm and emission window of 510–550 nm. To establish the range of the fully reduced or oxidized signal, cells were treated with 3 mM DTT or 5 mM H2O2 for 20 min. The ratio (R) of mean fluorescence from excitation at 405 and 488 nm was determined using Image J. Index of relative oxidation was calculated using equation IRO = 1-(R-RH2O2)/(RDTT- RH2O2).
Cells infected with retroviral vector pLPCX mt-roGFP2-Orp1 were visualized with a Zeiss LSM710 META confocal microscope (Carl Zeiss MicroImaging, Inc.) with excitation 405 at 488 nm and emission window of 510–550 nm. To establish the range of the fully reduced or oxidized signal, cells were treated with 3 mM DTT or 5 mM H2O2 for 20 min. The ratio (R) of mean fluorescence from excitation at 405 and 488 nm was determined using Image J. Index of relative oxidation was calculated using equation IRO = 1-(R-RH2O2)/(RDTT- RH2O2).