Intracellular ROS Detection Methods

Intracellular ROS amounts were detected using three different methods. CellROX® Green Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) detects total ROS intracellular content while MitoSOX^™ Red Indicator (Thermo Fisher Scientific, Waltham, Massachusetts, USA) specifically probes superoxide radicals. Detection was performed by immunofluorescence analysis. For immunofluorescence analysis, OVCAR3 and OVCAR8 cells were cultured on a cover slip, and upon 24 h, cells were incubated with CellROX® Green Reagent for 30 min. Both cell lines were incubated with MitoSOX^™ Red Indicator for 10 min at 37°C. Cells were then gently washed. Cover slips were mounted on microscope slides using a mounting solution ProLong Gold antifade reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Images were collected using a Leica DM-IRB/TC-SP2 confocal microscopy system (63x). ROS were also determined by incubating cells with the redox-sensitive probe 2′-7′-DCF (CM-H2CFDA; Molecular Probes, Eugene, OR, USA). Analysis was performed as described in Aversa et al. [26 (link)]. Fluorescence was revealed using the Victor3 Multilabel Counter (PerkinElmer, Turku, Finland) at 485 nm and 535 nm for excitation and emission, respectively. Results were normalized on protein concentration.