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Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Flow Cytometry*

Detection of intracellular superoxide in Jurkat cells using Cell Meter™ Fluorimetric Intracellular Superoxide Detection Kit. AMA Treatment (Red): Cells were treated with 50 µM Antimycin A (AMA) at 37 °C for 30 minutes, then incubated with MitoROS™ 580 for 1 hour. Control (Blue): Cells were incubated with MitoROS™ 580 at 37 °C for 1 hour without AMA treatment. The fluorescence signal was monitored at FL2 channel using a flow cytometer (BD FACSCalibur).
Detection of intracellular superoxide in Jurkat cells using Cell Meter™ Fluorimetric Intracellular Superoxide Detection Kit. AMA Treatment (Red): Cells were treated with 50 µM Antimycin A (AMA) at 37 °C for 30 minutes, then incubated with MitoROS™ 580 for 1 hour. Control (Blue): Cells were incubated with MitoROS™ 580 at 37 °C for 1 hour without AMA treatment. The fluorescence signal was monitored at FL2 channel using a flow cytometer (BD FACSCalibur).
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Catalog Number22970
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Telephone1-408-733-1055
Fax1-408-733-1304
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


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. Therefore, the detection of intracellular mitochondrial superoxide is of central importance to understanding proper cellular redox regulation and the impact of its dysregulation on various pathologies. Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit uses our unique superoxide indicator to quantify superoxide level in live cells. MitoROS™ 580 is live-cell permeant and can rapidly and selectively target superoxide in mitochondria. It generates red fluorescence when it reacts with superoxide. The Cell Meter™ Fluorimetric Intracellular Superoxide Detection Kit provides a sensitive, one-step fluorimetric assay to detect mitochondrial superoxide in live cells with one hour incubation. This kit is optimized for flow cytometry applications.

Platform


Flow cytometer

Excitation488 nm laser
Emission575/26 nm filter
Instrument specification(s)PE channel

Components


Component A: MitoROS™ 5801 vial
Component B: Assay Buffer1 bottle (10 mL)
Component C: DMSO1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells at a density of 0.5 - 1 × 106 cells/mL
  2. Treat the cells with test compounds to induce superoxide
  3. Add 1 µL 500X MitoROS™ 580 into 0.5 mL cell suspension
  4. Stain the cells at 37°C for 1 hour
  5. Monitor the fluorescence intensity using flow cytometer with FL2 channel

Important notes
Thaw all the components at room temperature before starting the experiment.

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™ 580 stock solution (500X):
Add 100 µL of DMSO (Component C) into the vial of MitoROS™ 580 (Component A) and mix well to make 500X MitoROS™ 580 stock solution. Protect from light. Note: For storage, seal tubes tightly.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 0.5 mL growth medium or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for superoxide induction.

  2. Treat cells with 25 µL of 20X test compounds in Assay Buffer (Component B) or your desired buffer (such as PBS) to induce superoxide. For control cells (untreated cells), add the corresponding amount of compound buffer.

  3. Incubate the cells at 37°C for at least 30 minutes or a desired period of time, protected from light. Note: Jurkat cells were treated with 50 µM Antimycin A (AMA) at 37°C for 30 minutes to induce superoxide. See Figure 1 for details. Pyocyanin (50 µM) or H2O2 (1 mM) can also be used to induce superoxide.

  4. Add 1 µL of 500X MitoROS™ 580 stock solution into 0.5 mL cell suspension.

  5. Incubate at 37°C for 1 hour. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with MitoROS™ 580. The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.

  6. Monitor the fluorescence intensity using a flow cytometer with FL2 channel (Ex/Em=488/590 nm). Gate on the cells of interest, excluding debris.

References


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Journal: J Endod (2014): 51
Effect of hypochlorite oxidation on cholinesterase-inhibition assay of acetonitrile extracts from fruits and vegetables for monitoring traces of organophosphate pesticides
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Journal: J Toxicol Sci (2014): 71
Green synthesis of carbon dots with down- and up-conversion fluorescent properties for sensitive detection of hypochlorite with a dual-readout assay
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Journal: Analyst (2013): 6551
Comparative antimicrobial activities of aerosolized sodium hypochlorite, chlorine dioxide, and electrochemically activated solutions evaluated using a novel standardized assay
Authors: Thorn RM, Robinson GM, Reynolds DM.
Journal: Antimicrob Agents Chemother (2013): 2216
Analysis of the germination kinetics of individual Bacillus subtilis spores treated with hydrogen peroxide or sodium hypochlorite
Authors: Setlow B, Yu J, Li YQ, Setlow P.
Journal: Lett Appl Microbiol (2013): 259
A simple yet effective chromogenic reagent for the rapid estimation of bromate and hypochlorite in drinking water
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Journal: Analyst (2013): 434
Effect of hypochlorite-based disinfectants on inactivation of murine norovirus and attempt to eliminate or prevent infection in mice by addition to drinking water
Authors: Takimoto K, Taharaguchi M, Sakai K, Takagi H, Tohya Y, Yamada YK.
Journal: Exp Anim (2013): 237
Use of pyrogallol red and pyranine as probes to evaluate antioxidant capacities towards hypochlorite
Authors: Perez-Cruz F, Cortes C, Atala E, Bohle P, Valenzuela F, Olea-Azar C, Speisky H, Aspee A, Lissi E, Lopez-Alarcon C, Bridi R.
Journal: Molecules (2013): 1638
Enhancement of anti-cholinesterase activity of aqueous samples by hypochlorite oxidation for monitoring traces of organophosphorus pesticides in water
Authors: Kanno A, Kawakami T, Takahashi Y, Onodera S.
Journal: J Toxicol Sci (2012): 389
Colorimetric determination of hypochlorite with unmodified gold nanoparticles through the oxidation of a stabilizer thiol compound
Authors: Zhang J, Wang X, Yang X.
Journal: Analyst (2012): 2806