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Mitosox red kit

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The MitoSOX Red kit is a fluorogenic dye designed to measure superoxide in the mitochondria of live cells. The dye selectively targets mitochondria and becomes fluorescent upon oxidation by superoxide.

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Lab products found in correlation

Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) Application suite x, by Leica camera (1 mentions) Cellquest, by BD (1 mentions) 6 well plate, by Thermo Fisher Scientific (1 mentions) Mitoprobe jc 1, by Thermo Fisher Scientific (1 mentions) Sml0737, by Merck Group (1 mentions) 510 confocal microscope, by Zeiss (1 mentions) 6 well tissue culture plate, by Corning (1 mentions) 12 well plate, by Thermo Fisher Scientific (1 mentions) Fluoromount media, by Merck Group (1 mentions) Fluorescence microscope, by Leica camera (1 mentions) Multimode plate reader, by Thermo Fisher Scientific (1 mentions) Anti flag antibody, by Merck Group (1 mentions) P h2ax ser139, by Cell Signaling Technology (1 mentions) Flag antibody conjugated agarose beads, by Merck Group (1 mentions) Premo autophagy tandem sensor rfp gfp lc3b kit, by Thermo Fisher Scientific (1 mentions) Dead cell apoptosis kit with annexin 5 fitc and pi for flow cytometry, by Thermo Fisher Scientific (1 mentions) Dm il led, by Leica camera (1 mentions) Sod typed assay kit hydroxylamine method, by Nanjing Jiancheng (1 mentions) Ros assay kit, by Beyotime (1 mentions)

16 protocols using mitosox red kit

1

Mitochondrial Superoxide Measurement

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Mitochondrial superoxide production was measured using the MitoSOX Red Kit (Invitrogen, Carlsbad, CA, USA). Briefly, mitochondria were isolated and 20 μg of mitochondria in 50 μl of respiration buffer was loaded into 96-well plate. One hundred microliters of ice cold respiration buffer was supplemented with 2 μM MitoSOX, 5 μM antimycin A and 4 μM ADP. Fifty micromoles salermide or 1 μM FeTCCP was added into indicated wells before loading into the FLUOstar Omega Microplate Reader. The basal level of fluorescence intensity was measured at 355 nm and 590 nm and fluorescence intensity was measured at 5-min intervals.
Chen T., Dai S.H., Li X., Luo P., Zhu J., Wang Y.H., Fei Z, & Jiang X.F. (2017). Sirt1-Sirt3 axis regulates human blood-brain barrier permeability in response to ischemia. Redox Biology, 14, 229-236.
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2

Mitochondrial Superoxide Assessment

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Mitochondrial superoxide levels were assessed with the MitoSOX Red Kit (Invitrogen, United States) (Zhang et al., 2019b (link)). In brief, 20 μg of isolated mitochondria was added to 50 μl of respiration buffer in 96-well plates. Then, 100 μl of chilled respiration buffer containing 2 μM MitoSOX, 5 μM antimycin A and 4 μM ADP were added. Finally, 50 μM salermide or 1 μM FeTCCP was supplemented to the indicated wells, followed by fluorescence reading every 5 min (background at 355 and 590 nm).
Zhu T., Zhu M., Qiu Y., Wu Z., Huang N., Wan G., Xu J., Song P., Wang S., Yin Y, & Li P. (2021). Puerarin Alleviates Vascular Cognitive Impairment in Vascular Dementia Rats. Frontiers in Behavioral Neuroscience, 15, 717008.
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3

MitoSOX Red Staining of Liver Macrophages

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Cells grown on coverslips were also stained with MitoSOX Red Kit (M36008, Invitrogen, China) (final concentration, 5µM) for 10 minutes in 37℃incubator, washed with PBS for three times, 5 minutes/each time. The primary liver macrophages and the PTPROt+ cells were then analyzed under an inverted confocal fluorescent microscope (Leica Microsystems CMS Gmbh Ernst-Leitz-Str, 17-37) and software (Leica Application Suite X, 3.6.20104.0) .
Jin K., Liu Y., Shi Y., Zhang H., Sun Y., Zhangyuan G., Wang F., Yu W., Wang J., Tao X., Chen X., Zhang W, & Sun B. (2020). PTPROt aggravates inflammation by enhancing NF-κB activation in liver macrophages during nonalcoholic steatohepatitis. Theranostics, 10(12), 5290-5304.
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4

Quantifying Mitochondrial Superoxide Levels

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The levels of mitochondrial superoxide (O2−) produced in the cells were quantified by using a MitoSOX Red kit (Molecular probe, Invitrogen), which contains a redox-sensitive dye with hydroethidine linked by a hexyl carbon chain to a triphenylphosphonium group, used to target the mitochondrial matrix due to the negative membrane potential across the inner membrane of mitochondria. The cells were plated 4 × 105 cells per well in 6-well plates (Nunc, Roskilde, Denmark). After treatment in hypoxic chamber, MitoSOX Red was added at a final concentration of 1.0 µM in HBSS (Gibco, Carlsbad, CA, USA). The cells were then incubated for 10 min at 37 °C, and then they were harvested and washed twice with PBS and finally added into 1 mL PBS. Identically, mitochondrial ROS was measured using FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using the flow cytometry analysis software BD CellQuest (Becton Dickinson, San Jose, CA, USA).
Kuo C.W., Tsai M.H., Lin T.K., Tiao M.M., Wang P.W., Chuang J.H., Chen S.D, & Liou C.W. (2017). mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells. International Journal of Molecular Sciences, 18(6), 1220.
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5

Mitochondrial ROS Quantification

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Mitochondrial ROS was measured using the MitoSOX Red kit (M36008 Molecular Probes) according to the manufacture's guide. In brief, cells were incubated with MitoSOX 5 µM in HBSS medium without FBS for 15 minutes in a CO2 incubator at 37ºC. Then cells were washed twice with warmed PBS, fluorescence was measured at Ex/Em: 510/580 nm and normalized to total protein. Mitochondrial ROS was also evaluated by fluorescence microscopy.
Barbier-Torres L., Chhimwal J., Kim S.Y., Ramani K., Robinson A., Yang H., Van Eyk J., Liangpunsakul S., Seki E., Mato J.M, & Lu S.C. (2024). S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver. International Journal of Biological Sciences, 20(4), 1218-1237.
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6

Mitochondrial ROS Quantification

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Mitochondrial ROS was measured using the MitoSOX Red kit (M36008 Molecular Probes) according to the manufacture’s guide. In brief, cells were incubated with MitoSOX 5 µM in HBSS medium without FBS for 15 min in a CO2 incubator at 37 °C. Then cells were washed twice with warmed PBS, fluorescence was measured at Ex/Em: 510/580 nm and normalized to total protein.
Barbier-Torres L., Murray B., Yang J.W., Wang J., Matsuda M., Robinson A., Binek A., Fan W., Fernández-Ramos D., Lopitz-Otsoa F., Luque-Urbano M., Millet O., Mavila N., Peng H., Ramani K., Gottlieb R., Sun Z., Liangpunsakul S., Seki E., Van Eyk J.E., Mato J.M, & Lu S.C. (2022). Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease. Nature Communications, 13, 557.
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7

Mitochondrial Membrane Potential and ROS

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Mitochondrial membrane potential was measured using the MitoProbe™ JC-1 (Molecular Probes, Eugene, OR) assay kit following the protocol provided. Mitochondrial ROS was measured using the MitoSOX™ Red kit (Molecular Probes, Eugene, OR) according to the manufacture’s guide. Five μM antimycin A was added 10 minutes after (SML0737, Sigma, St Louis, MO) or 5 μM of MitoTEMPO was added an hour prior to the start of the assay for positive and negative controls, respectively.
Murray B., Peng H., Barbier-Torres L., Robinson A., Li T.W., Fan W., Tomasi M.L., Gottlieb R.A., Eyk J.V., Lu Z., Martínez-Chantar M., Liangpunsakul S., Skill N.J., Mato J.M, & Lu S.C. (2019). Methionine Adenosyltransferase α1 is targeted to the mitochondrial matrix and interacts with cytochrome P450 2E1 to lower its expression. Hepatology (Baltimore, Md.), 70(6), 2018-2034.
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8

Measurement of Mitochondrial Superoxide

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ROS generation was measured using mitochondrial superoxide sensitive fluorophore MitoSOX Red kit (Molecular Probes, catalog no. M36008). The SVG-A and PHFA cells were grown in 6-well tissue culture plates (Corning, catalog no. 3506) and were transduced with either Ad-null or Ad-Agno WT (10 PFU/cell). At 18 h post-transduction, the transduced cells were transferred to 35-mm glass bottom poly-L-lysine coated plates (MatTek, catalog no. p35GC-14-C) and incubated for 5 hrs to allow the cell adhesion and were then treated with MitoSOX Red following the manufacturer’s recommendations. In brief, the culture medium was removed, and the transduced cells were treated with DMEM containing MitoSOX Red (5 uM) and counterstained with Hoechst 33342 (5 μg/ml) for 15 min 37°C in a humidified atmosphere with 5 % CO2. After staining, cells were washed twice with 1xPBS and the live cells were imaged using a Carl Zeiss 510 Confocal Microscope using a 40x oil objective at RFP/DAPI filter for MitoSOX Red and Hoechst respectively, while maintaining the cells at 37°C in a humidified atmosphere with 5 % CO2. Images were quantified using NIH ImageJ software (https://imagej.nih.gov/ij/). Mean integrated intensities of images (from 20 randomly chosen fields) in the red channel were determined after background subtraction and results were presented in graph forms.
Saxena R., Saribas S., Jadiya P., Tomar D., Kaminski R., Elrod J.W, & Safak M. (2020). Human Neurotropic Polyomavirus, JC Virus, Agnoprotein Targets Mitochondrion and Modulates Its Functions. Virology, 553, 135-153.
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9

Quantifying Mitochondrial Superoxide Levels

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The levels of mitochondrial superoxide (O2·−) produced in the cells were quantified using a MitoSOX Red kit (Molecular Probes, Invitrogen), which comprises a redox-sensitive dye composed of hydroethidine linked by a hexyl carbon chain to a triphenylphosphonium group, which was used to target the mitochondrial matrix due to the negative membrane potential across the inner mitochondrial membrane. The cells were plated at a density of 8 × 104 cells per well in 12-well plates (Nunc, Denmark) with a medium containing 25 mM glucose. MitoSOX Red was added at a final concentration of 1.0 μM in HBSS (Gibco BRL, USA). The cells were then incubated for 10 min at 37°C, and then they were harvested and washed twice with PBS. They were fixed in 4% paraformaldehyde and mounted in Fluoromount media (Sigma-Aldrich Co. LLC), which assisted in the visualization of the slides under a fluorescence microscope (Leica, Wetzlar, Germany). The average fluorescence intensity was quantitatively determined using ImageJ by counting 50–100 cells per field of view, five representative fields per experimental group, and three independent experiments.
Lin H.Y., Weng S.W., Chang Y.H., Su Y.J., Chang C.M., Tsai C.J., Shen F.C., Chuang J.H., Lin T.K., Liou C.W., Lin C.Y, & Wang P.W. (2018). The Causal Role of Mitochondrial Dynamics in Regulating Insulin Resistance in Diabetes: Link through Mitochondrial Reactive Oxygen Species. Oxidative Medicine and Cellular Longevity, 2018, 7514383.
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10

Quantifying Mitochondrial ROS in Cells

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Mitochondrial ROS level in cells was measured by the MitoSOX Red kit (Molecular Probes, Carlsbad, CA, USA) according to the manufacturer's procedures. Briefly, cells seeded in 96-well plates were incubated with MitoSOX Red probe at a final concentration of 5 μM for 10 min at 37 °C in the dark, and then gently washed three times by pre-warmed PBS before MF exposure. After exposure, fluorescence level was quantified by a multimode plate reader (Thermo Scientific, NY, USA) at excitation/emission wavelengths of 510 nm/580 nm. The number of experimental runs was 5, and in each experimental run, 5 replicate samples were used per experimental condition.
Feng B., Ye C., Qiu L., Chen L., Fu Y, & Sun W. (2016). Mitochondrial ROS Release and Subsequent Akt Activation Potentially Mediated the Anti-Apoptotic Effect of a 50-Hz Magnetic Field on FL Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 38(6).
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