In order to measure mitochondrial reactive oxygen species (ROS) levels, the cells were cultured in μ-dishes (ibidi, Munich, Germany) and then incubated with 5 μM MitoSOX Red (Thermo Fisher Scientific) and 1 μM Mitotracker Green (Thermo Fisher Scientific) for 30 min at 37 °C. Fluorescent images of MitoSOX Red and Mitotracker Green signals were acquired using a Nikon C2 confocal microscope (Nikon). The fluorescence intensity of MitoSOX Red and Mitotracker Green were measured using the NIS-Elements software (Nikon). The intensity of MitoSOX Red was divided by that of Mitotracker Green to calculate mitochondrial ROS production per mitochondrion.
Mitotracker green
Manufactured by Nikon
Sourced in Japan
MitoTracker Green is a fluorescent dye used to label mitochondria in live cells. It is a cell-permeant dye that stains mitochondria in a wide variety of cell types. The dye accumulates in active mitochondria, allowing for the visualization and tracking of mitochondrial structure and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker green, by Thermo Fisher Scientific (6 mentions)
μ dishes, by Ibidi (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Nis elements software, by Nikon (2 mentions)
C2 confocal microscope, by Nikon (2 mentions)
Mitosox red, by Nikon (2 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Nikon (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur instrument, by BD (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Mitotracker green, by Cell Signaling Technology (1 mentions)
Ax confocal microscope, by Nikon (1 mentions)
Mini26, by Merck Group (1 mentions)
7 protocols using mitotracker green
1
Measuring Mitochondrial Reactive Oxygen Species
2
Evaluating Mitochondrial Depolarization in MEFs
3
Mitochondrial Membrane Potential Measurement
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Isolated SMG acinar cells were loaded with 20 nM Tetramethylrhodamine, Ethyl Ester (TMRE; ThermoFisher Scientific: T669), and 1μM of MitoTracker Green (Invitrogen™; M7514). Fluorescence of both TMRE and MitoTracker Green was captured simultaneously using an inverted epifluorescence Nikon microscope with a 40 X oil immersion objective. The TMRE fluorescence was excited at 560 nm and emitted light collected at 574 nm; MitoTracker Green was excited at 488 nm and emitted light collected at 530 nm. Images were obtained every 1 s with an exposure of 20 ms and 4 × 4 binning using a digital camera controlled by TILL Photonics, TILLvision software. The acinar cells were exposed to 4 μM FCCP for 3 minutes by perfusion to rapidly dissipate the membrane potential. Mitochondrial membrane potential was quantified as the change in the ratio of TMRE/MitoTracker Green fluorescence before and after the administration of FCCP. Statistical analyses were performed with a t-test using Prism (GraphPad) as indicated in the figure legends.
4
Quantifying Cellular and Mitochondrial ROS
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To quantify cellular and mitochondrial reactive oxygen species (ROS) levels, cells were incubated with 5 μM CellROX Green (for cellular ROS; Thermo Fisher Scientific) or MitoSOX red (for mitochondrial ROS; Thermo Fisher Scientific) for 30 min. The cells were subsequently treated with TrypLE Express (Thermo Fisher Scientific) to detach them from the culture plate. The fluorescence signals of 10,000 cells were measured using a FACSCalibur instrument (BD Bioscience). The geometric means of the fluorescence signals were calculated using the Cell Quest software (BD Bioscience).
Mitochondrial ROS levels were determined using confocal microscopy as previously described.31 , 32 , 33 In brief, cells were cultured in μ‐dishes (Ibidi) and subsequently incubated with 5 μM MitoSOX red (Thermo Fisher Scientific) and 20 nM MitoTracker Green FM (Thermo Fisher Scientific) for 30 min. Fluorescent images of MitoSOX red and MitoTracker Green FM were acquired using a Nikon C2 confocal microscope. The fluorescence intensity of MitoSOX red and MitoTracker Green FM was measured using the NIS‐Elements software (Nikon). To determine mitochondrial ROS levels, the fluorescence intensities of MitoSOX red were divided by that of MitoTracker Green FM.
Mitochondrial ROS levels were determined using confocal microscopy as previously described.
5
Evaluating Apoptosis via Mitochondrial Membrane Potential
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The effects of AF on the cell mitochondrial membrane potential (Δψm) were examined by fluorescence microscope using JC-1 (Beyotime Biotech, Nantong, China) as specific probe. Cells were treated with AF for 14 h and stained with JC-1 in a humidified atmosphere of 5% CO2 at 37°C for 30 minutes. Images acquired from monomer and aggregate were merged and viewed under the Nikon fluorescence microscope (40X amplification, Nikon, Japan). Evaluation of the sub-cellular localization of cytochrome C was done by using fluorescence imaging of cells double-labeled with MitoTracker Green (Molecular Probes) and cytochrome C antibody. After treatment, cells were incubated with 100 nM MitoTracker Green, fixed with 3% paraformaldehyde, permeabilized with 0.02% Triton X and blocked with 5% BSA, followed by treatment with primary rabbit polyclonal cytochrome C antibody for 2 h at room temperature and Cy2-conjugated goat anti-rabbit antibody for 1 h. Cellular images were acquired by using a fluorescence microscope (40X amplification, Nikon, Japan).
6
Multiparametric Analysis of Cellular Redox State
7
Labeling and Imaging of EVs and Mitochondria
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The RPC‐EVs were labelled with PKH26 following the manufacturer's instructions (MINI26; Sigma‐Aldrich). Briefly, EV pellets were resuspended in 1 mL diluent C before mixing with the stain solution (4 µL PKH26 Cell Linker in ethanol and 1 mL diluent C). After staining for 5 min at room temperature, 2 mL of 1% bovine serum albumin was added to the mixture, which was then diluted to 70 mL with PBS, followed by ultracentrifugation at 100,000 × g for 70 min. The EV pellets were then resuspended in PBS and were used for subsequent experiments. The same labelling procedure without EVs was performed as the negative control. According to the manufacturer's instructions, ARPE‐19 cells were incubated with MitoTracker Green (9074; Cell Signaling Technology, USA), which was added to the medium at a 100 nM concentration for 30 min to stain mitochondria. Images were captured at 561 nm (PKH26) and 488 nm (MitoTracker Green) using a confocal laser scanning microscope (AX confocal microscope; Nikon, Japan).
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