Mitosox

Manufactured by BD

Sourced in United States

MitoSOX is a fluorogenic dye that can be used to measure superoxide in the mitochondria of live cells. It is a cell-permeable, positively charged dye that selectively targets the mitochondria and becomes fluorescent upon oxidation by superoxide.

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Lab products found in correlation

Mitosox, by Thermo Fisher Scientific (25 mentions) Mitosox red, by Thermo Fisher Scientific (7 mentions) Facscalibur flow cytometer, by BD (6 mentions) H2dcfda, by Thermo Fisher Scientific (5 mentions) H2dcfda, by BD (3 mentions) Facscalibur, by BD (3 mentions) Facscanto 2, by BD (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Tcs sp5 confocal scanning microscope, by Leica camera (2 mentions) Facsdiva software, by BD (2 mentions) Facscanto flow cytometer, by BD (2 mentions) Cm h2dcfda, by Thermo Fisher Scientific (2 mentions) Axio imager widefield fluorescence microscope, by Zeiss (2 mentions) Facscanto 2 flow cytometry, by BD (2 mentions) Total ros detection kit, by Enzo Life Sciences (2 mentions) Mitosox, by Enzo Life Sciences (2 mentions) Color matched beads, by BD (2 mentions) Tom20 antibodies, by Novus Biologicals (2 mentions) Cd66b clone g10f5, by BioLegend (2 mentions) Dnase, by Roche (2 mentions)

34 protocols using mitosox

Mitochondrial ROS Quantification by Flow Cytometry

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To monitor the accumulation of mitochondrial ROS, cells were stained with the mitochondria-specific superoxide sensor MitoSOX (Thermo Fisher Scientific). In brief, cells were washed once and then stained with 1 μM of MitoSOX in Hanks' balanced salt solution for 10 minutes at 37°C. After washing, dead cells were stained using SYTOX Blue dead cell stain (Thermo Fisher Scientific). The percentage of MitoSOX-positive cells and the mean fluorescence intensity of MitoSOX were quantified by flow cytometry (BD LSRII) and normalized to the mean fluorescence intensity of the solvent control probe.

Gomez Solsona B., Horn H., Schmitt A., Xu W., Bucher P., Heinrich A., Kalmbach S., Kreienkamp N., Franke M., Wimmers F., Schuhknecht L., Rosenwald A., Zampieri M., Ott G., Lenz G., Schulze-Osthoff K, & Hailfinger S. (2023). Inhibition of glutaminase-1 in DLBCL potentiates venetoclax-induced antitumor activity by promoting oxidative stress. Blood Advances, 7(24), 7433-7444.

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Mitochondrial Reactive Species Detection

Cited 1 time

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Reactive species-detecting probe MitoSOX Red (Molecular Probes, Invitrogen, Eugene, OR, USA) (50 nM, 37 °C, 30 min) was interacting with MitoSOX to generate fluorescence, which was determined by a flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) [30 (link)].

Peng S.Y., Wang Y.Y., Lan T.H., Lin L.C., Yuan S.S., Tang J.Y, & Chang H.W. (2020). Low Dose Combined Treatment with Ultraviolet-C and Withaferin a Enhances Selective Killing of Oral Cancer Cells. Antioxidants, 9(11), 1120.

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Mitochondrial ROS Measurement in Neutrophils

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For measurement of mitochondrial ROS (mtROS) release, 1 × 10⁶ neutrophils were incubated with 10 μM rotenone (Tocris Bioscience, Bristol, UK), 200 μm MitoTEMPO (Cayman Chemical, Ann Arbor, MI, USA), 10 μM TX or TX plus rotenone and loaded with 2.5 μM MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 30 min. After incubation, MitoSOX™ fluorescence intensity was measured using a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) at wavelengths of 510 nm excitation and 580 nm emission.

Salinas C., Barriga K., Albornoz A., Alarcon P., Quiroga J., Uberti B., Sarmiento J., Henriquez C., Ehrenfeld P., Burgos R.A, & Moran G. (2023). Tamoxifen triggers the in vitro release of neutrophil extracellular traps in healthy horses. Frontiers in Veterinary Science, 9, 1025249.

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Quantifying Mitochondrial Superoxide

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Cells were stained with the mitochondrial superoxide indicator MitoSOX (Invitrogen) as described in the manufacturer's protocols. Cells were loaded with 5 μM MitoSOX for 30 min and washed three times with PBS. MitoSOX fluorescence were measured using a FACSCalibur (Becton-Dickinson, San Diego, CA) and were gated and analysed using forward and side-scatter plots on 10,000 live events.

Kim B.R., Kim B.J., Kook Y.H, & Kim B.J. (2020). Mycobacterium abscessus infection leads to enhanced production of type 1 interferon and NLRP3 inflammasome activation in murine macrophages via mitochondrial oxidative stress. PLoS Pathogens, 16(3), e1008294.

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Quantifying Cellular and Mitochondrial ROS

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DCFH-DA (2′,7′-dichlorofluorescein diacetate; Sigma-Aldrich, St. Louis, MO) and MitoSOX Red (Molecular Probes, Eugene, OR, USA) were used to measure cellular ROS and mitochondrial superoxide, respectively. Cells were incubated with 50 μm DCFH-DA or 5 μm MitoSOX at 37 °C for 30 min. The emitted DCF and MitoSOX fluorescence were quantified using a FACSCalibur flow cytometer (Becton-Dickinson) with excitation/emission wavelengths of 485/530 nm to measure DCF in the FL1 channel and with excitation/emission wavelengths of 510/580 nm to measure oxidized MitoSOX Red in the FL2 channel.

Tseng H.Y., Chen Y.A., Jen J., Shen P.C., Chen L.M., Lin T.D., Wang Y.C, & Hsu H.L. (2017). Oncogenic MCT-1 activation promotes YY1-EGFR-MnSOD signaling and tumor progression. Oncogenesis, 6(4), e313-.

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Measuring Cellular Oxidative Stress

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H9c2 cells seeded on glass coverslips were loaded with the ROS-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen, CA, USA; 2.5 μmol L⁻¹) or the mitochondrial

O_{2}^{\cdot -}

-specific fluorescent probe MitoSOX (Invitrogen; 3 μmol L⁻¹) - dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v) – which were added to cell culture media for 15 min at 37 °C, as described^{18 (link)}. The cells were fixed in 2% buffered paraformaldehyde for 10 min at room temperature and the H₂DCFDA and MitoSOX fluorescence analysed using a Leica TCS SP5 confocal scanning microscope equipped with an argon laser source (excitation λ 488 nm or 543 nm, respectively) and a x63 oil immersion objective. ROS and mitochondrial

O_{2}^{\cdot -}

generation were also monitored by flow cytometry^{54 (link)}: briefly, single-cell suspensions were incubated with H₂DCFDA (1 μmol L⁻¹) or MitoSOX (0.5 μmol L⁻¹) for 15 min at 37 °C and immediately analysed using a FACSCanto flow cytometer (Becton–Dickinson). Data were analyzed using FACSDiva software (Becton–Dickinson).

Becatti M., Bencini A., Nistri S., Conti L., Fabbrini M.G., Lucarini L., Ghini V., Severi M., Fiorillo C., Giorgi C., Sorace L., Valtancoli B, & Bani D. (2019). Different Antioxidant Efficacy of Two MnII-Containing Superoxide Anion Scavengers on Hypoxia/Reoxygenation-Exposed Cardiac Muscle Cells. Scientific Reports, 9, 10320.

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Intracellular and Mitochondrial ROS Measurement

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2′,7′-Dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich) and MitoSOX red (Molecular Probes, Invitrogen, Carlsbad, CA, USA) were used to measure the levels of intracellular ROS and mitochondrial ROS, respectively, as described previously [50 (link)]. Cells were pretreated with NAC (100 µM) or Mito-TEMPO (50 µM) for 30 min, followed by the treatment with either the indicated concentrations of ABN-B for 2 h or 30 µM of ABN-B for the indicated period of time. The cells were then treated with either DCF-DA (10 µM) or MitoSOX (5 µM) in the dark for 30 min, and harvested after washing with PBS. Their fluorescence intensities were assessed using a FACS Calibur flow cytometer (Becton-Dickinson, for DCF-DA, excitation wavelength: 485 nm, emission wavelength: 535 nm; for MitoSOX, excitation wavelength: 510 nm, emission wavelength: 580 nm).

Phan T.N., Kim O., Ha M.T., Hwangbo C., Min B.S, & Lee J.H. (2020). Albanol B from Mulberries Exerts Anti-Cancer Effect through Mitochondria ROS Production in Lung Cancer Cells and Suppresses In Vivo Tumor Growth. International Journal of Molecular Sciences, 21(24), 9502.

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Mitochondrial ROS Quantification in Light-Damaged Retinas

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Mitochondrial ROS levels in light-damaged retinas were assessed using the intracellular mitochondrial superoxide probe, MitoSox (Molecular probes, Life Technologies). Single cell suspensions were loaded with 5 µM MitoSox for 15 min at 37 °C, and fluorescence was measured using a Becton-Dickinson FACScan flow cytometer. MitoSox Red was excited by laser at 488 nm, and the data collected at FSC, SSC, 670LP (FL3) channel. 10,000 events were counted in the gated photoreceptor region. Cell debris, as represented by distinct low forward and side scatter, were gated out for analysis. Results were analyzed using FlowJo software (Trustees of Leland Stanford Jr. University).

Byrne A.M., Ruiz-Lopez A.M., Roche S.L., Moloney J.N., Wyse -Jackson A.C, & Cotter T.G. (2016). The synthetic progestin norgestrel modulates Nrf2 signaling and acts as an antioxidant in a model of retinal degeneration. Redox Biology, 10, 128-139.

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Quantifying Oxidative Stress in Bronchial Cells

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ROS were detected using the general and mitochondria-specific oxidative sensitive fluorescent dyes CM-H₂DCFDA (ThermoFisher) and MitoSOX (ThermoFisher), respectively. For flow cytometric analysis, human bronchial epithelial cell line (NHBEs, ATCC^® CRL-4051™) were treated with 50 µg/mL CRE in the presence or absence of either the antioxidant N-acetyl cysteine (1 to 10 mM) (Sigma) or the mitochondria-specific antioxidant MitoTEMPO (1 to 10 nM) (Sigma) 1 hour prior to treatment in F-12K media at 37°C in a 5% CO₂ atmosphere. ROS production was assessed using a FACScalibur flow cytometer (Becton Dickinson Biosciences) for detection of CM-H₂DCFDA and MitoSOX. Under the same conditions, ROS production in mitochondria was assessed with MitoSOX by fluorescence microscopy. In brief, cells were incubated with 5 µM MitoSOX and 0.1 µM MitoTracker (ThermoFisher) for 30 min prior to CRE treatment. These cells were then imaged with a Nikon ETi fluorescence microscope. Fluorescence intensities were quantified using ImageJ (NIH).

Qiu L., Zhang Y., Do D.C., Ke X., Zhang S., Lambert K., Kumar S., Hu C., Zhou Y., Ishmael F.T, & Gao P. (2018). miR-155 Modulates Cockroach Allergen and Oxidative Stress-Induced Cyclooxygenase-2 in Asthma. Journal of immunology (Baltimore, Md. : 1950), 201(3), 916-929.

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Detecting Cellular ROS and Mitochondrial Superoxide

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The ROS-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA, 2.5 μmol L⁻¹, Invitrogen, CA, USA) or the mitochondrial O₂^•−-specific fluorescent probe MitoSOX (3 μmol L⁻¹, Invitrogen) dissolved in 0.1% DMSO and 0.01% w/v Pluronic acid F-127 were added to H9c2 cells seeded on glass coverslips for 15 min at 37 °C, as previously described [13 (link)]. Cells were fixed at room temperature for 10 min in 2% buffered paraformaldehyde and fluorescence analyzed using a Leica TCS SP8 confocal scanning microscope equipped with an argon laser source (excitation λ 488 nm or 543 nm, respectively) and a 63× oil immersion objective. ROS and mitochondrial O₂^•− production were also estimated by flow cytometry [21 (link)]: briefly, single-cell suspensions were incubated with H₂DCFDA (1 μmol L⁻¹) or MitoSOX (0.5 μmol L⁻¹) for 15 min at 37 °C, and immediately analyzed under a FACSCantoII flow cytometer (Becton–Dickinson) [22 (link)]. Data were analyzed using FACSDiva software (Becton Dickinson, San Jose, CA, USA).

Nistri S., Fiorillo C., Becatti M, & Bani D. (2020). Human Relaxin-2 (Serelaxin) Attenuates Oxidative Stress in Cardiac Muscle Cells Exposed In Vitro to Hypoxia–Reoxygenation. Evidence for the Involvement of Reduced Glutathione Up-Regulation. Antioxidants, 9(9), 774.

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