Intracellular O2•- was determined independently either by a lucigenin-based chemiluminescence assay or by flow cytometry using O2•--sensitive fluorescent probe, dihydroethidium (DHE) (Thermo Fisher Scientific, Massachusetts, USA). For lucigenin-based assay, harvested cells were washed with 1X PBS and lysed in 450 μl of ATP lysis buffer (Sigma Aldrich, Missouri, USA). Upon cell lysis, lysates were immediately aspirated for chemiluminescence monitoring using the Berthold Sirius Luminometer (Bad Wildbad, Germany) at a rate of 0.6 s interval over 14.4 s. The luminescence emission was measured and recorded as relative light units (RLU). The average of RLUs was then calculated and subjected to normalization by respective sample protein concentration, determined by Coomassie Plus protein assay reagent (Pierce Biotechnology, Massachusetts, USA).
For flow cytometry-based detection of O2•- harvested cells were resuspended with plain medium containing 5 μM DHE and incubated for 15 min at 37 °C. Stained cells were then analysed by CytoFLEX LX Flow Cytometer (Beckman Coulter, California, USA) with excitation wavelength set at 595 nm and cell counter at 10,000 events. Subsequent data analysis was done using the CytExpert software.
For flow cytometry-based detection of O2•- harvested cells were resuspended with plain medium containing 5 μM DHE and incubated for 15 min at 37 °C. Stained cells were then analysed by CytoFLEX LX Flow Cytometer (Beckman Coulter, California, USA) with excitation wavelength set at 595 nm and cell counter at 10,000 events. Subsequent data analysis was done using the CytExpert software.