Mitosox

Dynamic analysis of cellular redox was carried out using four different live cell imaging dyes, that detect specific molecular species: cytoplasmic superoxide is analysed using DHE (Life Technologies, D11347); mitochondrial superoxide is detected with mitoSOX™ Red (Life Technologies, M36008); global cellular hydrogen peroxide (H₂O₂) and peroxyl radicals (HO₂) are measured using DCFDA (Sigma, D6883) while unbound reduced glutathione (GSH) is assayed with MCB (Life Technologies, M-1381MP). Cells were cells co-transfected with an appropriate fluorophore that would not interfere with the fluorescence of the dye. All dyes were administered to the recording medium (RM) onto cells plated on coverslips, within Attafluor® metal cell chambers (Molecular Probes™, Thermo fisher, A-7816). RM composition is: 5.6 mMKCl (Sigma, P9333), 10 mM D-(+)-Glucose (Sigma, G7528), 10 mM HEPES (Sigma, H4304), 4.2 mM NaHCO₃ (Sigma, 56297), 138 mM NaCl (Sigma, S5886), 2.6 mM CaCl₂ (Sigma, C7902), 1.2 mM NaH₂PO₄ (BDH,1024940), 1.2 mM MgCl₂ (BDH, 10149), pH 7.4.
DHE (10 μM), mitoSOX (10 μM), and DCFDA (15 μM) were diluted in RM and imaged onto a Zeiss LSM510 confocal microscope. MCB was used at final concentration of 2.5 μM in RM. Brightness controlling settings were maintained consistently within the experiment for all techniques.

Intracellular ROS production was quantified by measuring the fluorescence intensities of DHE (Thermo Fisher Scientific) and MitoSOX (Thermo Fisher Scientific) as ROS indicators. Frozen liver tissues were sectioned with 10 μm thickness. Tissue sections were then washed in phosphate-buffered saline (PBS) and incubated with 20 μM DHE and 5μM MitoSOX at 37°C for 30 min. The DHE and MitoSOX stained cell areas were quantified using a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany).

Cells were seeded in 6-well plates, and then photosensitization experiments (MB 10 μM, laser 40 J/cm²) were performed after 48 h when the cells had grown to 80-90% confluence. Six hours after MB-PDT treatment, the cells were stained with 20 μM DCFH-DA (YEASEN, Shanghai, China) to measure intracellular ROS, such as hydroxyl radicals and peroxynitrite anion, and 1 μM MitoSOX (YEASEN, Shanghai, China) to measure mitochondrial ROS (superoxides) for 30 minutes at 37°C in a humidified atmosphere in the dark [26 (link), 27 (link)]. The cells were then gently washed twice with phosphate-buffered saline (PBS). Both intracellular and mitochondrial ROS were identified using a fluorescence spectrophotometer (Zeiss, Gottingen, Germany). Both DCF (green fluorescence, 488 nm excitation wavelength) and MitoSOX (red fluorescence, 555 nm excitation wavelength) florescence intensities were analyzed using Zeiss CLSM software (ZEN 2009 Light Edition). The experiments were repeated three times independently.

HEK293T cells were treated with hydrogen peroxide (H2O2) (Sigma-Aldrich, 216763) at a concentration of 500 μM for 2 h, alone or in combination with 5 mM N-acetyl-cyteine (NAC) (Sigma-Aldrich, A7250). When using NAC, cells were pre-treated for 2hrs with 5 mM NAC. The mitochondrial superoxide indicator stain MitoSOX (ThermoFisher, M36008) was used to probe the relative oxidative stress in live cells. Cells were stained with 1uM MitoSOX diluted in DMEM. 250,000 cells were incubated with 330 ul for 30 min and analyzed by flow cytometry, then washed with PBS and trypsinized. Flow cytometry tubes were kept on ice and in the dark until use. Flow cytometry analysis was performed with a FACSCalibur (BD Biosciences). The mean fluorescence intensity of minimum 10,000 stained cells and unstained control cells were recorded and plotted for analysis. Alternatively, MitoSOX was analyzed by epifluorescence microscopy (Zeiss, Axio Observer).

Denuded oocytes were fixed using acetic acid:ethanol (1:3) solution overnight at RT and then were stained with 2% orcein solution for 10 min. The meiotic maturation stage of each oocyte was confirmed under a microscope (Leica).
Denuded oocytes were incubated in IVM medium with 5 μM dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen) or 1 μM Mito-SOX red (Invitrogen) for 30 min at 38.5°C. The fluorescence images of DCF-DA and Mito-SOX were acquired using an LSM 800 confocal microscope (Zeiss).