The effects of cobalt chloride exposure on mitochondrial reactive oxygen species (ROS) production and apoptosis were measured in N2A cells using MitoSOX™ Mitochondrial Superoxide Indicator (ThermoFisher Scientific Waltham, MA, USA) and Incucyte® Caspase‐3/7 Green Dye (Essen BioScience Ltd., Hertfordshire, UK), respectively, according to manufacturers’ instructions. The dyes were diluted in serum‐free culture medium with 300 μM of cobalt chloride and added to cells plated on a PDL‐coated 96‐well plate. Images from live cells were acquired immediately and after every hour using IncuCyte® S3 Live Cell Analysis System (Essen BioScience Ltd., Hertfordshire, UK) red (MitoSOX™) and green (Caspase‐3/7) channels. Phase contrast images were used to count the total number of cells and the percentage of the red and green cells.
Mitosox mitochondrial superoxide indicator
Manufactured by Thermo Fisher Scientific
Sourced in United States
MitoSOX mitochondrial superoxide indicator is a fluorogenic dye that is highly selective for superoxide in the mitochondria of live cells. It is a useful tool for detecting mitochondrial oxidative stress.
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Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Incucyte caspase 3 7 green dye, by Sartorius (1 mentions)
Incucyte s3 live cell analysis system, by Sartorius (1 mentions)
Facsverse cytometer, by BD (1 mentions)
Mt20 illumination system, by Olympus (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Varioskan lux multimode plate reader, by Thermo Fisher Scientific (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Ab133303, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
M36008, by Thermo Fisher Scientific (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Cd25 pe, by Thermo Fisher Scientific (1 mentions)
Cd11b pe cy5, by Thermo Fisher Scientific (1 mentions)
Cd3 pecy5, by Thermo Fisher Scientific (1 mentions)
Foxp3 pe cy5, by Thermo Fisher Scientific (1 mentions)
25 protocols using mitosox mitochondrial superoxide indicator
1
Cobalt Chloride-Induced Mitochondrial ROS and Apoptosis
2
Mitochondrial ROS Generation Analysis
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The generation of ROS by mitochondria was analyzed using the MitoSOX mitochondrial superoxide indicator (Thermo Fisher Scientific). HaCaT cells were incubated with R1-MVs (30 μg/mL) or ΔDR2577-R1-MVs (30 μg/mL) for 12 h prior to treatment with H2O2 (0.3 mM) for 12 h at 37 °C The HaCaT cells were cultured with 5 μM MitoSOX reagent for 10 min at 37 °C in the dark, washed, and resuspended in PBS. The samples were analyzed using a FACSverse cytometer and FlowJo software (V10, BD Biosciences).
3
Measuring Mitochondrial Superoxide in Mouse Cardiomyocytes
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Adult mouse ventricular myocytes were stained with 5 μM MitoSOX mitochondrial superoxide indicator (M36008, Thermo fisher) in Tyrode buffer containing (in mM): 140 NaCl, 5.4 KCl, 2 MgCl2, 1.8 CaCl2, 10 HEPES, and 10 glucose (pH 7.4 at 37 °C) at 37 °C for 15 min and washed twice buffer before imaging. Cells were excited at 550 nm and the emitted light was recorded at 570 nm using the IX83 microscope and the MT20 illumination system (Olympus). Light intensity was set to 4% and the exposure time was 500 ms. Each image was recorded at an interval of 120 s. After the fluorescence intensity stabilised as baseline readings, DMSO or ouabain at different concentrations was added, respectively. Cells were then washed with Tyrode buffer to remove ouabain. The Tyrode solution used for imaging contained 10 μM Blebbistatin. After the measurement, fluorescent data were extracted by the cellSens software. The fluorescent intensities of each cell at different time points were normalised against the mean baseline of its own. The measurement was repeated three times on different days with different isolations of ventricular cardiomyocytes from mice with 3–4 months of age independently.
4
Quantification of Blastocyst ROS Levels
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Total ROS in blastocysts were detected using 2′,7′-dichlorodihydrofluorescein diacetate (D399; Molecular Probes, Eugene, OR, USA) as previously described [43 (link), 44 ]. Briefly, blastocysts were incubated for 15 min in PBS and PVA containing 10 µM 2′,7′‐dichlorodihydrofluorescein diacetate at 37 °C. After incubation, the blastocysts were washed three times with PBS and PVA. Fluorescence signals were captured as a Tagged Image File Format (TIFF) file using a digital camera (DP72; Olympus, Tokyo, Japan) connected to a fluorescence microscope (IX70; Olympus). The MitoSox Mitochondrial Superoxide Indicator (Thermo Fisher Scientific) was used to evaluate the generation of mitochondrial ROS. Briefly, blastocysts were incubated for 30 min in PZM-5 containing 10 µM MitoSox solution at 37 °C. After incubation, blastocysts were washed three times with PBS and PVA and fixed in 3.7% paraformaldehyde for 30 min at room temperature. Total and mitochondria-derived ROS levels were quantified by analyzing the fluorescence intensity in blastocysts using ImageJ v.l.44 g software (National Institutes of Health, Bethesda, MD, USA).
5
Mitochondrial ROS Detection in Blastocysts
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Mitochondrial ROS were detected using MitoSOX Mitochondrial Superoxide Indicator (Thermo Fisher Scientific), as previously described (Nasr-Esfahani et al., 1990 (link); Lee and Hyun, 2017 (link)). Briefly, ten blastocysts each group were treated with 10 µM MitoSOX indicator in the IVC medium for 30 min. Subsequently, the blastocysts were washed thrice with PBS/PVA and fixed in 3.7% paraformaldehyde for 30 min in the dark. TOM20 was stained according to the instruction in the immunofluorescence and confocal microscopy subsection. The fluorescence intensity of the mitochondria-derived ROS was analyzed using the ImageJ v. 44 g software (National Institutes of Health, Bethesda, MD, United States).
6
Quantifying Intracellular and Mitochondrial ROS
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Intracellular ROS levels were measured by staining the cells with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo Fisher Scientific). The cells were treated with 10 µM H2DCFDA for 30 min at 37 °C in the dark and then washed with PBS. Mitochondrial ROS levels were measured using MitoSOX mitochondrial superoxide indicator (Thermo Fisher Scientific). The cells were stained with 5 µM MitoSOX reagent for 10 min at 37 °C in the dark and then washed with PBS. The samples were immediately analyzed using the FACSVerseTM flow cytometer, and data were processed using FlowJo software.
7
Mitochondrial Stress and DNA Damage
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The expression of γH2AX (DNA double-strand break marker protein) and levels of MitoSOX and Rhod-2-AM were examined using immunofluorescence. In brief, cells on coverslips, were treated with H2O2 and morroniside as described above, and then probed with γH2AX antibody (Cat. No. BLR053F, Thermo Fisher Scientific), MitoSOX™ Mitochondrial Superoxide Indicator (Cat. No. M36008, Thermo Fisher Scientific), and Rhod-2 AM, fluorescent mitochondrial Ca2+ indicator (Cat. No. ab142780, Abcam, Inc.) in accordance with each manufacturer’s recommendations (Duan et al., 2023 (link)).
8
Evaluating Mitochondrial Function in TDSCs
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ROS Assay Kit (Beyotime, China) and MitoSox Mitochondrial Superoxide Indicator (Thermo Scientific, USA) were used to detect ROS and superoxide of TDSCs according to instructions. And JC‐1 (Beyotime, China) and Mito‐Tracker Green/Red CMxRos (Yeasen, China) were used to detect the mitochondrial membrane potential of TDSCs. Briefly, after TDSCs were exposed to 100 × 10−6 m H2O2 in serum‐free α‐MEM for 4 h, the probe was mixed with serum‐free culture medium and diluted to work concentration. After incubating for 20 min, the Hoechst staining solution (1:100, Servicebio, China) was used to stain the nuclei. Then the cell plates were observed and the images were captured using DMi8 fluorescent microscopy. The cellular ATP levels were determined luminometrically using ATP assay reagent (Beyotime, China) according to previously mentioned protocol.[40 ] Briefly, the TDSCs lysates were collected and used to determine ATP content. The resulting luminescence was measured by Varioskan LUX multimode plate reader (Thermo Scientific, USA). Finally, ATP concentrations were calculated according to a standard curve.
9
Quantifying Blastocyst ROS Levels
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Total ROS levels in blastocysts were determined using 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCF‐DA, Cat # D399, Molecular Probes, Eugene, OR, USA) as previously described.17 ,6 Briefly, blastocysts were incubated for 15 minutes in phosphate‐buffered saline (PBS)/PVA containing 10 µmol/L H2DCF‐DA at 38.5°C. After incubation, blastocysts were washed three times with PBS/PVA. Fluorescence signals were captured as a TIFF file using a digital camera (DP72, Olympus, Tokyo, Japan) connected to a fluorescence microscope (IX70, Olympus). MitoSOX™ mitochondrial superoxide indicator (Thermo Fisher) was then used to evaluate the generation of mitochondrial ROS. Briefly, blastocysts were incubated for 30 minutes in PZM‐5 containing 10 µmol/L MitoSOX™ solution at 38.5°C. After incubation, the blastocysts were washed three times with PBS/PVA and fixed in 3.7% (v/v) paraformaldehyde for 30 minutes at room temperature (20‐25°C). Total and mitochondria‐derived ROS levels were quantified by analysing the fluorescence intensity of the blastocysts using Image J version 1.44g software (National Institutes of Health, Bethesda, MD, USA).
10
Fluorescent Imaging of NOX4, IL-1β, and Mitochondrial Activity
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Fluorescence staining of lung tissue and BEAS-2B cells was performed. Briefly, primary rabbit monoclonal antibody against NOX4 (ab133303, Abcam) used for incubation overnight at 4°C, followed by incubation with AlexaFluor488 goat anti-rabbit (A11001, Life Technologies, Waltham, MA, USA) for 2 h. BEAS-2B cells were stained with primary antibody against IL-1β (12703, CST) overnight at 4°C and then with AlexaFluor488 goat anti-rabbit (A11001, Life Technologies) for 2 h. Mitochondrial membrane potential (MMP) fluorescence staining was performed using tetramethylrhodamineethylester (TMRE) (113852, Abcam) at a final concentration of 50 nM at 37°C for 30 min. The mtROS fluorescence staining was performed using MitoSOX (M36008, mitoSOX mitochondrial superoxide Indicator, Thermo Fisher) at a final concentration of 5 μM at 37°C for 10 min. All data were collected by Cytation ™ 5 (BioTek Instruments).
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