Mitosox red indicator

Mitochondrial superoxide was determined using the MitoSOX Red indicator (M36008, Thermo Fisher Scientific). NRVMs were seeded into a black 96-well plate (clear bottom with lid) and repeatedly exposed to aconitine (0, 2.5, 5, 10, 20, 40, and 80 μM, respectively) or DA (50 μM) for 7 days. After treatment, cells were washed with PBS buffer and then incubated with 5 μM MitoSOX Red for 10 min at 37°C. Next, fluorescence intensities of each group were detected with a multilabel microplate reader (Victor X5, PerkinElmer, Waltham, MA, United States), and results were presented as mean ± SD.

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.

The MitoSOX Red indicator (Thermo Fisher Scientific, USA) was used to evaluate mtROS in cells treated for one month. A cell monolayer was incubated with 2.5 µM MitoSOX in Hank’s Balanced Salt Solution (1X) for 15 min. At least 10,000 events were recorded in a Cytek Aurora CS cytometer using PE—Red (Ex/Em: 510/580 nm) channel. 100 µM CoCl₂ (Sigma-Aldrich, USA) was used as a positive control according to previous reports^{11 (link)}. Treatment with CoCl₂ failed to promote cell death (data no show).

Cells at 60% to 70% confluence were treated with 800M H₂O₂ for 24h. A total of 1% FBS was added to avoid severe cell injury, which is optimal to determine the protective effects of the intervention. The cell apoptosis was determined with an AnnexinV-APC/PI apoptosis detection kit (Sony Biotechnology, 3804660) according to the manufacturers instructions. Cell reactive oxygen species (ROS) were determined by the Total Reactive Oxygen Species Assay Kit (Thermo Fisher, 88-5930-74). Mitochondrial superoxide was stained with MitoSOX red indicator (Thermo Fisher, M36008). Mitochondrial permeability transition pore (mPTP) opening was determined with the MitoProbe Transition Pore Assay Kit (Thermo Fisher, M34153).

All forms of DMEM, mitoSOX red indicator and rat tail collagen I were purchased from Life Technologies (Paisley, UK). HepG2 cells were purchased from European Collection of Cell Cultures (ECACC, Salisbury, UK). NAD⁺/NADH ratio assay kit was purchased from eEnzyme (Maryland, USA). Cytotoxicity detection kits were purchased from Roche Diagnostics Ltd (West Sussex, UK). Clear and white 96-well plates were purchased from Fisher Scientific (Loughborough, UK) and Greiner Bio-One (Stonehouse, UK) respectively. All OCR consumables were purchased from Seahorse Bioscience (North Billerica, Massachusetts, USA). All other reagents and chemicals were purchased from Sigma Aldrich (Dorset, UK).

Mitochondrial O₂⁻ production was measured using MitoSOX Red indicator (Life Technologies) as described (Tronchere et al., 2017 (link)). Briefly, cells were loaded with MitoSOX (2 µm) for 30min, washed in PBS, PFA-fixed, mounted on glass slide using MOWIOL and imaged on a Zeiss LSM780 confocal microscope. Frozen heart cryosections were hydrated in PBS and incubated with MitoSOX (5 µm) for 30 min at 37°C in a humidified chamber, quickly washed and imaged on a Zeiss LSM780 confocal microscope. The MitoSOX fluorescence intensity was quantified using Image J software on thresholded images.

The cells were seeded onto 24-mm glass coverslips and allowed to grow to 60–70% confluence. The cells were then incubated with MitoSOX-Red indicator (Life Technologies Ltd) for 30 min and washed. When indicated, cells were treated with the pro-apoptotic compound menadione. After washing, the amount of mitochondrial superoxide was determined using a Tali™ image-based cytometer (Life Technologies). The data are presented as a histogram showing the % of the mean intensity of MitoSOX fluorescence.