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Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit *Green Fluorescence*

Mitochondria are major producers of cellular superoxide. The production of low to moderate levels of superoxide is critical for the proper regulation of many essential cellular processes including gene expression, signal transduction, and muscle adaptation to endurance exercise training. Uncontrolled mitochondrial superoxide production can trigger cellular oxidative damage that contributes to the pathogenesis of a wide variety of disorders including cancer, cardiovascular diseases, neurodegenerative diseases and aging. The detection of intracellular mitochondrial superoxide is of great importance to understanding proper cellular redox regulation and the impact of its dysregulation on various pathologies. Cell Meter™ Fluorimetric Intracellular Superoxide Detection Kit uses MitoROS™ 520, our unique superoxide indicator, to quantify superoxide level in live cells. MitoROS™ 520 is cell permeant and can rapidly and selectively detect superoxide in mitochondria. It generates green fluorescence upon reacting with superoxide. The Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit provides a sensitive, one-step fluorimetric assay to detect mitochondrial superoxide in live cells with one hour incubation. This kit can be used for flow cytometry and fluorescence microscopy applications.

Example protocol

AT A GLANCE

Protocol summary (Fluorescence Microscope)

  1. Prepare cells in growth medium
  2. Treat the cells with test compounds to induce superoxide
  3. Add MitoROS™ 520 working solution
  4. Incubate the cells at 37°C for 1 hour
  5. Monitor the fluorescence using FITC fliter set

Protocol summary (Flow Cytometry)

  1. Prepare cells in growth medium
  2. Treat the cells with test compounds to induce superoxide
  3. Add MitoROS™ 520 stock solution and incubate the cells at 37°C for 1 hour
  4. Monitor the fluorescence intensity with a flow cytometer using 530/30 nm filter (FITC channel)

Important notes
Thaw all the components at room temperature before use.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. MitoROS™ 520 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ 520 (Component A) and mix well. Note: 25 µL of reconstituted MitoROS™ 520 stock solution is enough for 1 plate. Note: Unused portion can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

Only for Fluorescence Microscope
Add 5 μL of 500X DMSO reconstituted MitoROS™ 520 stock solution into 2 mL of Assay Buffer (Component B) and mix well. Note: This working solution is not stable and needs to be prepared freshly before use.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

For Fluorescence Microscopes/96-Well Microplates:

  1. Treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in medium or your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of compound buffer.

  2. To induce superoxide, incubate the cell at 37°C for a desired period of time, protected from light. Note: We treated RAW 264.7 macrophage cells with 5 µM Antimycin A (AMA) at 37°C for 2 hours to induce superoxide. See Figure 1 for details.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ 520 working solution into the cell plate.

  4. Incubate the cells at 37°C for 1 hour, and take images using fluorescence microscope with a FITC filter set.

For Flow Cytometers:

  1. Treat cells as desired.

  2. To induce superoxide, incubate the cells at 37°C for a desired period of time, protected from light. Note: We treated Jurkat cells with 50 µM Antimycin A (AMA) at 37°C for 2 hours to induce superoxide.

  3. Add 1 µL/0.5 mL cells of MitoROS™ 520 stock solution (500X) into the cells.

  4. Incubate the cells in a 5% CO2, 37°C incubator for 1 hour, and monitor the fluorescence intensity using a flow cytometry with 530/30 nm filter (FITC channel).

Spectrum

Citations

View all 3 citations: Citation Explorer
Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics
Authors: Akinci, Ersin and Cha, Minsun and Lin, Lin and Yeo, Grace and Hamilton, Marisa C and Donahue, Callie J and Bermudez-Cabrera, Heysol C and Zanetti, Larissa C and Chen, Maggie and Barkal, Sammy A and others,
Journal: BioRxiv (2020)
Homogeneously catalytic oxidation of phenanthrene by the reaction of extracellular secretions of pyocyanin and Nicotinamide Adenine Dinucleotide
Authors: Nie, Hongyun and Nie, Maiqian and Diwu, Zhenjun and Wang, Lei and Qiao, Qi and Zhang, Bo and Yang, Xuefu
Journal: Environmental Research (2020): 110159
Comparison of the detrimental features of microglia and infiltrated macrophages in traumatic brain injury: A study using a hypnotic bromovalerylurea
Authors: Abe, Naoki and Choudhury, Mohammed E and Watanabe, Minori and Kawasaki, Shun and Nishihara, Tasuku and Yano, Hajime and Matsumoto, Shirabe and Kunieda, Takehiro and Kumon, Yoshiaki and Yorozuya, Toshihiro and others, undefined
Journal: Glia (2018)

References

View all 52 references: Citation Explorer
Mitochondrial flashes: dump superoxide and dance with protons now
Authors: Demaurex N, Schwarzl and er M., undefined
Journal: Antioxid Redox Signal. (2016)
Up-Regulation of Mitochondrial Antioxidant Superoxide Dismutase Underpins Persistent Cardiac Nutritional-Preconditioning by Long Chain n-3 Polyunsaturated Fatty Acids in the Rat
Authors: Abdukeyum GG, Owen AJ, Larkin TA, McLennan PL.
Journal: J Clin Med (2016): 32
Expression and polymorphism (rs4880) of mitochondrial superoxide dismutase (SOD2) and asparaginase induced hepatotoxicity in adult patients with acute lymphoblastic leukemia
Authors: Alachkar H, Fulton N, Sanford B, Malnassy G, Mutonga M, Larson RA, Bloomfield CD, Marcucci G, Nakamura Y, Stock W.
Journal: Pharmacogenomics J. (2016)
Adipocyte-Specific Deletion of Manganese Superoxide Dismutase Protects From Diet-Induced Obesity Through Increased Mitochondrial Uncoupling and Biogenesis
Authors: Han YH, Buffolo M, Pires KM, Pei S, Scherer PE, Boudina S.
Journal: Diabetes (2016): 2639
Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling
Authors: Br, undefined and MD., undefined
Journal: Free Radic Biol Med. (2016)
Page updated on December 6, 2024

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Catalog Number16060
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Spectral properties

Excitation (nm)

513

Emission (nm)

537

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Flow cytometer

Excitation488 nm laser
Emission530, 30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall, clear bottom
Instrument specification(s)FITC filter set

Components

Fluorescence images of superoxide measurement in macrophage cells using cat#16060. RAW 264.7 cells at 100,000 cells/well/100 &micro;L were seeded overnight in a 96-well black wall/clear bottom plate. AMA Treatment: Cells were treated with 5 &micro;M Antimycin A (AMA) at 37 &deg;C for 2 hours, then incubated with MitoROS&trade; 520 for 1 hour. Untreated Control: RAW 264.7 cells were incubated with MitoROS&trade; 520 at 37 &deg;C for 1 hour without AMA treatment. The fluorescence signal was measured using fluorescence microscope with a FITC filter
Fluorescence images of superoxide measurement in macrophage cells using cat#16060. RAW 264.7 cells at 100,000 cells/well/100 &micro;L were seeded overnight in a 96-well black wall/clear bottom plate. AMA Treatment: Cells were treated with 5 &micro;M Antimycin A (AMA) at 37 &deg;C for 2 hours, then incubated with MitoROS&trade; 520 for 1 hour. Untreated Control: RAW 264.7 cells were incubated with MitoROS&trade; 520 at 37 &deg;C for 1 hour without AMA treatment. The fluorescence signal was measured using fluorescence microscope with a FITC filter
Fluorescence images of superoxide measurement in macrophage cells using cat#16060. RAW 264.7 cells at 100,000 cells/well/100 &micro;L were seeded overnight in a 96-well black wall/clear bottom plate. AMA Treatment: Cells were treated with 5 &micro;M Antimycin A (AMA) at 37 &deg;C for 2 hours, then incubated with MitoROS&trade; 520 for 1 hour. Untreated Control: RAW 264.7 cells were incubated with MitoROS&trade; 520 at 37 &deg;C for 1 hour without AMA treatment. The fluorescence signal was measured using fluorescence microscope with a FITC filter
Fluorescence images of endogenous superoxide measurement in macrophage cells using Cat#16060. RAW 264.7 cells at 100,000 cells/well/100 &micro;L were seeded overnight in a 96-well black wall/clear bottom plate. LPS Treatment: Cells were incubated with MitoROS&trade; 520 for 1 hour, then treated with 200 &mu;g/mL of lipopolysaccharide (LPS) at 37 &ordm;C for 16 hours. Untreated Control: RAW 264.7 cells were incubated with MitoROS&trade; 520 at 37 &ordm;C for 1 hour without LPS treatment. The fluorescence signal was measured using fluorescence microscope with a FITC filter.