ROS generation within mitochondrial compartment was assessed in live cells using MitoSOX Red, a fluorogenic dye that is taken up by mitochondria where it is readily oxidized by superoxide anion and serves as mitochondrial ROS indicator. Briefly, neurons were seeded in 6-well culture plates, and MitoSOX Red (Beyotime Institute of Biotechnology, Shanghai, China) was dissolved in DMSO to form 5 mM stock solution. After treatment, neurons were loaded with 1 μM MitoSOX Red for 10 min at 37°C in the dark. Then, neurons were washed with PBS, and fluorescence intensity was determined with fluorescence microplate reader at 510 nm excitation and 580 nm emission, respectively. Data were recorded as folds of increased fluorescence intensity in treated neurons compared with that of untreated neurons.
Mitosox red
Manufactured by Beyotime
Sourced in China
MitoSOX Red is a fluorogenic dye used to detect superoxide in the mitochondria of live cells. It is a cell-permeant dye that selectively targets the mitochondria and exhibits red fluorescence upon oxidation by superoxide.
Automatically generated - may contain errors
Lab products found in correlation
Mito tracker green, by Beyotime (4 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Beyotime (2 mentions)
Confocal laser scanning microscope, by Leica camera (1 mentions)
Jc 1 assay kit, by Beyotime (1 mentions)
Automatic microplate reader, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Beyotime (1 mentions)
Jc 1 fluorescent probe, by Beyotime (1 mentions)
Srt 1720, by Beyotime (1 mentions)
H 9500, by Hitachi (1 mentions)
Mitotracker red, by Beyotime (1 mentions)
Mitotracker, by Beyotime (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Facscanto, by BD (1 mentions)
C11 bodipy, by Thermo Fisher Scientific (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Beyotime (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Cm h2dcfda, by Beyotime (1 mentions)
Lsm 800, by Zeiss (1 mentions)
11 protocols using mitosox red
1
Quantifying Mitochondrial ROS Generation
2
Mitochondrial ROS and Membrane Potential Assay
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The levels of mitochondrial ROS were assayed by MitoSOX™ Red (Red, Thermo Fisher, Waltham, MA, USA). Briefly, the RMECs were stained with the MitoSOX™ Red (5 μM, 10 min) and then co-stained with MitoTracker-Green (200 nM, Green, 30 min, Beyotime, Nantong, China). The mitochondrial ROS was analyzed by laser scanning confocal microscopy (40×, Leica, Weztlar, Germany).
On the other hand, MMP was assessed by a JC-1 assay kit (Beyotime, Nantong, China). Briefly, the RMEC cells were incubated with JC-1 staining solution. The cells were visualized under the laser scanning confocal microscope (40×, Leica, Weztlar, Germany). MMP was calculated as the ratio of red/green using ImageJ software.
On the other hand, MMP was assessed by a JC-1 assay kit (Beyotime, Nantong, China). Briefly, the RMEC cells were incubated with JC-1 staining solution. The cells were visualized under the laser scanning confocal microscope (40×, Leica, Weztlar, Germany). MMP was calculated as the ratio of red/green using ImageJ software.
3
Quantifying Cellular Oxidative Stress
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ROS/RNS production was assessed using the ROS-sensitive dyes 2′,7′-dichloro-dihydrofluorescein diacetate (DCFH-DA) (Beyotime Biotechnology), MitoSox Red (Beyotime Biotechnology) and 3-Amino,4-aminomethyl-2′,7′ -difluorescein (DAF-FM DA) (Beyotime Biotechnology). Cells or worms were treated with 20 μM DCFH-DA in Hank's Balanced Salt Solution (HBSS) for 30 min at 37 °C, 1 μM MitoSox Red in HBSS for 20 min at 37 °C, or 0.5 mM DAF-FM DA in its buffer solution. The samples were then washed three times with PBS and immediately measured using an automatic microplate reader (Thermo). The excitation and emission wavelengths were 485 and 535 nm, respectively, for the DCFH-DA fluorescence intensity, 510 and 580 nm for MitoSox Red, and 495 and 515 nm for DAF-FM DA.
4
Evaluating Mitochondrial Dysfunction in NP Cells
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According to the manufacturer's instructions, the MitoTracker Green (Beyotime, C1048, China), MitoSOX-Red (Beyotime, C1045, China), and JC-1 fluorescent probe (Beyotime, C2006, China) were used to detect mitochondrial membrane potential changes in NP cells. The morphology of mitochondria of NP cells was observed by transmission electron microscopy (TEM; H-9500; Hitachi, Tokyo, Japan). The decrease in mitochondrial membrane potential is a hallmark event in the early stages of apoptosis [76 ,77 (link)]. JC-1 (5,5′,6,6′-tetrachloro1,1′,3,3′-tetramethylbenzimidazolylcarbocyanine iodide) is an ideal fluorescent probe widely used to detect mitochondrial membrane potential (△Ψm). JC-1 monomers can produce green fluorescence (△Ψm↓) while JC-1 aggregates can produce red fluorescence (△Ψm↑) [78 ,79 (link)]. We measured the proportion of mitochondrial depolarization by comparing the relative proportions of red and green fluorescence. Therefore, the transition of JC-1 from red fluorescence to green fluorescence can detect the decrease of cell membrane potential and serve as an early detection indicator of apoptosis. Then we used the selective SIRT1 activator (SRT-1720, Beyotime, SC0267) and the selective SIRT1 inhibitor (EX-527, MCE, HY-15452) to verify the mechanism of PEVs repairing impaired mitochondria in pathological NP cells. All experiments were performed with four replicates each.
5
Mitochondrial ROS Detection in HK-2 Cells
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Intercellular mitochondrial ROS production in HK-2 cells was detected using the fluorescent reagent MitoSOX Red (M36008, Invitrogen, Carlsbad, CA, United States). Briefly, after treatment, HK-2 cells were incubated with MitoSOX Red (5 μM) and MitoTracker green (50 nM, C1048, Beyotime, China) at 37°C for 20 min in the dark, or the treated cells were incubated with DCFH-DA (10 μM) and MitoTracker Red (50 nM) at 37°C for 30 min in the dark. For kidney tissue, 4-μm-thick frozen sections were stained with 5 μM MitoSOX Red at 37°C for 20 min in the dark, followed by washing three times with PBS. The images were analyzed under a fluorescence microscope.
6
Cellular Autophagy and Oxidative Stress Assay
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A total of 1 × 105 cells were seeded in six-well plates containing cell slides. Transfection was performed after 24 h. Before fixation, DMSO was added for 8 h, followed by CQ for 8 h and CCCP for 4 h. DCFH-DA, MitoSOX Red, MitoPY1, and MitoTracker (1:1000, C1032, Beyotime) were added for 10–30 mins. After 24 h, cells were fixed with 4% formaldehyde for 20 mins at room temperature. Subsequently, the cells were washed three times with PBS and incubated with buffer containing 2% BSA and 0.3% Triton X-100 for 1 hr at room temperature. Then, the cells were incubated with the corresponding primary antibody at 4°C for 4 h. After three washes with PBS, the cells were incubated with the secondary antibody for 2 h at room temperature. Following another wash with PBS, the cells were incubated with DAPI (1:1000) for 5 mins at room temperature (Note: Some cells may skip this step and not incubate with DAPI). Finally, the cells were sealed, air-dried overnight, and then photographed using a confocal microscope with a 20× or 60× oil lens.
7
Measuring Intracellular and Mitochondrial ROS
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The intracellular and mitochondrial ROS levels were quantified by measuring the fluorescence of DCFH-DA-A (cat. no. S0033; Beyotime Institute of Biotechnology) and MitoSOX™ Red (Mitochondrial Superoxide Indicator; cat. no. M36008; Thermo Fisher Scientific, Inc.), respectively. After the aforementioned treatments, H9c2 cells were collected and washed three times with 1X PBS, followed by incubation with 5 µM DCFH-DA-A or MitoSOX™ Red for 45 min at 37°C in the dark. H9c2 cells were then washed three times with 1X PBS and detached with trypsin/EDTA. The relative level of cellular fluorescence was quantified by flow cytometry (BD FACSCanto™; BD Biosciences). Data were analyzed using FlowJo version ×0.7 (FlowJo LLC).
8
Lipid Peroxidation and Mitochondrial ROS Imaging
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The probes of lipid peroxidation probe BODIPY-C11 (Thermo Fisher Scientific, USA) and mtROS probe MitoSOX (Thermo Fisher Scientific, USA) were used in TM4 cells. Hoechst (Beyotime, China) was used to stain TM4 cell nuclei. TM4 cells were treated with C11-BODIPY for 30 min and washed with PBS. TM4 cell was incubated with Mito Tracker Green (Beyotime, China) for 30 min, and incubated in MitoSOX Red for 10 min. The signal was analyzed using a fluorescence microscope (Leica, Germany).
9
Fluorescent Probes for Intracellular H2O2 and Mitochondrial O2- Imaging
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DCFH-DA (Sigma), a H2O2-sensitive fluorescence probe, was applied to assess intracellular H2O2 levels, as previously described. MitoSOX Red (Invitrogen), a live-cell-permeant dye that rapidly and selectively targets mitochondria, was used to observe mitochondrial O2-. Briefly, cells were loaded with 10 µM DCFH-DA for 30 min at 37 °C. The cells were then incubated with 5 µM MitoSOX Red for 10 min at 37 °C, and the nuclei were stained with Hoechst 33342 (Beyotime) for 5 min. The DCFH-DA fluorescence and MitoSOX Red fluorescence were visualized using a laser confocal microscope (LeicaTCS SP5II, Germany).
10
Oxidative Stress-Induced ROS Detection
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After exposure to 50 µM H2O2 for 1 h, the HEI-OC1 cells were cultured using the ROS Assay Kit (S0033S; Beyotime Institute of Biotechnology), added together with an appropriate volume of diluted 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; 1:1,000), and then inoculated to a 6-well plate. Then cells were separately dyed by Mito-SOX Red (C1049-50 µg; Beyotime Institute of Biotechnology) staining with a final concentration of 4 µmol/l at 37˚C for 10 min in the dark or CM-H2DCFDA (S0033S; Beyotime Institute of Biotechnology) staining at a final concentration of 5 µmol/l at 37˚C for 30 min in the dark. After washing three times with PBS (C0221A; Beyotime Institute of Biotechnology), the cells were captured by laser confocal microscope (LSM800; Zeiss) at different excitation wavelengths (510 nm/488 nm) and different emission wavelengths (580/515 nm), and fluorescence images were captured.
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