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Mitosox staining

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MitoSOX is a fluorogenic dye used for detecting superoxide in the mitochondria of live cells. It selectively targets the mitochondria and becomes fluorescent upon oxidation.

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Lab products found in correlation

Ros glo h2o2 assay kit, by Promega (2 mentions) Tcs sp8 sted, by Leica (1 mentions) Laser confocal microscope, by Nikon (1 mentions) A1rmp confocal microscope, by Nikon (1 mentions) Atp bioluminescent assay kit, by Abcam (1 mentions) Mitotracker red cmxros probe, by Thermo Fisher Scientific (1 mentions) Standard assay kits, by Beyotime (1 mentions) Dcfh da staining, by Beyotime (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) Fluostar omega plate reader, by BMG Labtech (1 mentions) 710 nlo, by Zeiss (1 mentions) Polarstar optima plate reader, by BMG Labtech (1 mentions) Nucblue live cell stain, by Thermo Fisher Scientific (1 mentions) Tumor necrosis factor (tnf), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

MitoSOX staining kits MitoSOX Red Staining Kit MitoSOX red staining MitoSOX

8 protocols using mitosox staining

1

Mitochondrial Superoxide Quantification

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Mitochondrial superoxide levels were determined using analysis of fluorescent microscopy images and fluorometer readings of MitoSOX staining (Thermo Fisher Scientific). For fluorescent images, transduced Pan02 cells were seeded at 1 x 105 cells per mL in a 24 well plate and allowed to incubate overnight. The following day, media was replaced with experimental media of complete media +/- 10 ng/mL TNF (PeproTech) and incubated for 24 hours. MitoSOX staining (Thermo Fisher Scientific) was done according to manufacturer’s protocol and cells were counterstained with NucBlue Live Cell Stain (Thermo Fisher Scientific). Fiji-ImageJ was used to split the fluorescent channels, remove background, and measure mean gray value. For the fluorometer readings, transduced Pan02 cells were seeded at 1 x 104 cells per well in a 96 well plate and allowed to incubate for 48 hours in complete media. Media was then replaced with experimental media of complete media +/- 100 ng/mL TNF (PeproTech), +/- 45% glucose for one hour. MitoSOX staining (Thermo Fisher Scientific) was done according to manufacturer’s protocol and cells were counterstained with NucBlue Live Cell Stain (Thermo Fisher Scientific). Fluorescence was measured using a fluorometer, and RFP (MitoSOX) fluorescence was corrected for DAPI (NucBlue) fluorescence to normalize superoxide levels to the number of cells per well.
Nagai-Singer M.A., Morrison H.A., Woolls M.K., Leedy K., Imran K.M., Tupik J.D, & Allen I.C. (2023). NLRX1 functions as a tumor suppressor in Pan02 pancreatic cancer cells. Frontiers in Oncology, 13, 1155831.
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2

Mitochondrial ROS Evaluation in Heart

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To evaluate mitochondrial reactive oxygen species (ROS) levels, the heart tissues were prepared into frozen sections for MitoSOX staining (Invitrogen). Fluorescence images were captured using a Leica laser scanning confocal microscope (LSCM, TCS SP8 STED; Leica), and images were processed using Image‐Pro Plus 6.0 Software.
Jiang J., Liang S., Zhang J., Du Z., Xu Q., Duan J, & Sun Z. (2020). Melatonin ameliorates PM2.5‐induced cardiac perivascular fibrosis through regulating mitochondrial redox homeostasis. Journal of Pineal Research, 70(1), e12686.
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3

Quantifying Tissue ROS and Mitochondrial ROS

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To evaluate reactive oxygen species (ROS) and mitochondrial ROS levels, the aorta tissues were prepared into frozen sections for DHE staining (BBoxiProbe, China) and MitoSOX staining (Invitrogen, USA) according to the manufacturer's protocols. Then sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were acquired using a confocal laser microscope (Nikon, Japan) and processed by Image J software.
Ning R., Li Y., Du Z., Li T., Sun Q., Lin L., Xu Q., Duan J, & Sun Z. (2021). The mitochondria-targeted antioxidant MitoQ attenuated PM2.5-induced vascular fibrosis via regulating mitophagy. Redox Biology, 46, 102113.
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4

Quantifying Mitochondrial Dynamics in Cardiomyocytes

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The mitochondria in the primary cardiomyocytes were labeled with MitoTracker Red CMXRos probe (100 mmol/L, Invitrogen, Carlsbad, USA) and imaged using a confocal laser-scanning microscope (Nikon A1R MP + Confocal Microscope). The number and volume of the mitochondria observed were analyzed and quantified as previously described [17 ]. After MitoTracker Red staining, flow cytometry was performed to analyze mitochondrial mass. MitoSOX staining (Invitrogen, Carlsbad, USA) was performed to evaluate mitochondrial ROS levels in cardiomyocytes. Intracellular ATP content was detected using an ATP bioluminescent assay kit (Biovision, California, USA) according to the manufacturer's protocol.
Liu C., Han Y., Gu X., Li M., Du Y., Feng N., Li J., Zhang S., Maslov L.N., Wang G., Pei J., Fu F, & Ding M. (2021). Paeonol promotes Opa1-mediated mitochondrial fusion via activating the CK2α-Stat3 pathway in diabetic cardiomyopathy. Redox Biology, 46, 102098.
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5

Quantifying Mitochondrial ROS in VPCs

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Mitochondrial reactive oxygen species (ROS) in VPCs were examined by Mito‐Sox staining (Invitrogen, M36008). Briefly, VPCs were seeded in 24‐well plates with glass coverslips. After different treatments, VPCs were washed with PBS and incubated with 5 μmol/L Mito‐sox at 37 °C for 15 minutes in the dark. Subsequently, the VPCs from different treatments were randomly photographed. Images of five different view fields for each slide were captured randomly by a motorized inverted microscope (Olympus, Hamburg, Germany) at 535‐nm excitation and 610‐nm emission wavelengths. The fluorescence intensity was analysed using Image J software (National Institutes of Health, Bethesda, MD, USA) in three independent experiments. The level of mitochondrial ROS was normalized by the fluorescence intensity of control cells.
He H., Yu B., Liu Z., Ye G., You W., Hong Y., Lian Q., Zhang Y, & Li X. (2019). Vascular progenitor cell senescence in patients with Marfan syndrome. Journal of Cellular and Molecular Medicine, 23(6), 4139-4152.
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6

Apoptosis and Oxidative Stress Quantification

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TUNEL labeling (Roche, Shanghai, China) and caspase-3 activity colorimetric assay kits (KeyGEN Biotech, Jiangsu, China) were used to determine cellular apoptosis as described previously [18 (link)]. The apoptosis index was obtained by dividing the number of TUNEL-positive apoptotic cells by the total number of nucleated cells stained with 4′,6-diamino-2-phenylindole (DAPI). The caspase 3 colorimetric assay kit is based on the hydrolysis of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA) moiety. The p-NA produces a yellow color and can be quantified using a spectrophotometer or a microtiter plate reader reading absorbance at 405 nm. DCFH-DA staining (Beyotime Biotechnology, Nantong, China) and MitoSOX staining (Invitrogen, Carlsbad, USA) were used to measure cellular and mitochondrial ROS levels in the cardiomyocytes. DCFH was added to the cardiac homogenate to evaluate ROS production, and the fluorescence was measured using a fluorometer [19 (link)]. Total superoxide dismutase (SOD), cellular CuZnSOD, mitochondrial MnSOD, and malondialdehyde (MDA) levels were determined utilizing standard assay kits (Beyotime Biotechnology, Nantong, China) following the manufacturer's recommendations.
Ding M., Shi R., Cheng S., Li M., De D., Liu C., Gu X., Li J., Zhang S., Jia M., Fan R., Pei J, & Fu F. (2022). Mfn2-mediated mitochondrial fusion alleviates doxorubicin-induced cardiotoxicity with enhancing its anticancer activity through metabolic switch. Redox Biology, 52, 102311.
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7

Quantifying Cellular Hydrogen Peroxide Levels

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Cells were seeded in a 96-well plate and treated under various conditions. Cellular levels of H2O2 were determined using the ROS-Glo H2O2 Assay Kit (Promega). Briefly, derivatized luciferin substrate from the kit was incubated with samples, reacting directly with H2O2 to generate the luciferin precursor. After the addition of the ROS-Glo Detection Solution, which converts the luciferin precursor to luciferin, the luminescence signal was recorded by a FLUOstar Omega Plate Reader (BMG Labtech). The ROS luminescence signal was normalized to protein quantification. Mitochondrial ROS levels were estimated by MitoSOX Staining (Thermo Fisher Scientific). Briefly, cells were incubated with 5 μmol/L MitoSOX-Red for 30 minutes at 37°C. The fluorescence signal was assessed on a FACSCanto II Flow Cytometer (BD Biosciences), and fluorescence intensity was quantified using ImageJ (version 1.52K, NIH, Bethesda, MD).
Liu Y., Pang Y., Zhu B., Uher O., Caisova V., Huynh T.T., Taieb D., Vanova K.H., Ghayee H.K., Neuzil J., Levine M., Yang C, & Pacak K. (2020). Therapeutic Targeting of SDHB-Mutated Pheochromocytoma/Paraganglioma with Pharmacologic Ascorbic Acid. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(14), 3868-3880.
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8

Measurement of Cellular and Mitochondrial ROS

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Cellular ROS was measured using ROS-Glo H2O2 assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The luminescence signal was measured using Polarstar Optima plate reader (BMG LABTECH, Ortenberg, Germany). Mitochondrial ROS were measured using MitoSOX staining (Thermo Fisher). Cells were incubated with 5 μM MitoSOX-Red at 37 °C for 10 min and analyzed by either confocal imaging using Zeiss 710 NLO or flow cytometry using LSRFortessa SORP or FACSCanto II. Fluorescence intensity was measured and quantified by using ImageJ software (v1.8.0_112).
Liu Y., Pang Y., Caisova V., Ding J., Yu D., Zhou Y., Huynh T.T., Ghayee H., Pacak K, & Yang C. (2020). Targeting NRF2-Governed Glutathione Synthesis for SDHB-Mutated Pheochromocytoma and Paraganglioma. Cancers, 12(2), 280.
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