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Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Orange Fluorescence*

Images of HeLa cells stained with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit in a Costar black wall/clear bottom 96-well plate. A: Untreated control cells. B: Cells treated with 100 µM tert-butyl hydroperoxide (TBHP) for 30 minutes before staining.
Images of HeLa cells stained with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit in a Costar black wall/clear bottom 96-well plate. A: Untreated control cells. B: Cells treated with 100 µM tert-butyl hydroperoxide (TBHP) for 30 minutes before staining.
Detection of ROS in Hela cells. Hela cells were seeded overnight at 15,000 cells/90µL/well in a Costar black wall/clear bottom 96-well plate. The cells were untreated (control) or treated with1 mM H<sub>2</sub>O<sub>2 </sub>or 100 µM tert-butyl hydroperoxide (TBHP) for 30min at 37 °C. The ROS Brite™ 570 assay solution (100µL/well) was added and incubated in a 5% CO<sub>2</sub>, 37 °C incubator for 1 hour. The fluorescence signal were monitored at Ex/Em = 540/570 nm (Cutoff= 550 nm) with bottom read mode using FlexStation (Molecular Devices).
Detection of ROS in Jurkat cells. Jurkat cells were treated without (Green) or with 100µM tert-butyl hydroperoxide (TBHP) (Red) for 30min at 37 °C, and then loaded with ROS Brite™ 570 in a 5% CO<sub>2</sub>, 37 °C incubator for 1 hour. The fluorescence intensities were measured with Acea flow cytometer using PE channel.
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Catalog Number22902
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Reactive oxygen species (ROS) are natural byproducts of the normal metabolism of oxygen and play important roles in cell signaling. The accumulation of ROS results in significant damage to cell structures. The role of oxidative stress in cardiovascular disease, diabetes, osteoporosis, stroke, inflammatory diseases, a number of neurodegenerative diseases and cancer has been well established. The ROS measurement will help to determine how oxidative stress modulates varied intracellular pathways. Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit uses our proprietary ROS Brite™ 570 sensor to quantify ROS in live cells. The cell-permeable and non-fluorescent ROS Brite™ 570 exhibits a strong fluorescence signal upon reaction with ROS. ROS Brite™ 570 sensor is localized in the cytoplasm. The fluorescence signal of ROS Brite™ 570 sensor can be measured by fluorescence microscopy, high-content imaging, microplate fluorometry, or flow cytometry. The Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit provides a sensitive, one-step fluorimetric assay to detect intracellular ROS (especially superoxide and hydroxyl radical) in live cells within 1 hour incubation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format using either a fluorescence microplate reader or a fluorescent microscope with TRITC filter.

Platform


Flow cytometer

Excitation488 nm or 532 nm laser
Emission575/26 nm filter
Instrument specification(s)PE channel

Fluorescence microscope

ExcitationTRITC filter
EmissionTRITC filter
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation540 nm
Emission570 nm
Cutoff550 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: ROS Brite™ 5701 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: DMSO1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol A summary (Fluorescence microplate reader, fluorescence microscope)

  1. Prepare cells in growth medium
  2. Treat the cells with test compounds to induce ROS
  3. Add ROS Brite™ 570 working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
  4. Stain the cells at 37 °C for 30 - 60 minutes
  5. Monitor the fluorescence increase (bottom read mode) at Ex/Em= 540/570 nm (Cutoff = 550 nm) or fluorescence microscope with TRITC filter set 

Protocol B summary (Flow cytometer)

  1. Prepare cells in growth medium
  2. Treat cells with test compounds to induce ROS
  3. Incubate ROS Brite™ 570 with the cells for 30 - 60 minutes
  4. Monitor the fluorescence intensities using flow cytometer with FL2 channel

Important notes
Thaw all the kit 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. ROS Brite™ 570 stock solution (500X):
Add 40 µL of DMSO (Component C) into the vial of ROS Brite™ 570 (Component A) and mix well to make 500X ROS Brite™ 570 stock solution. Protect from light. Note: 20 µL of 500X ROS Brite™ 570 stock solution is enough for 1 plate. For flow cytometer, 500X ROS Brite™ 570 stock solution can be diluted by 5 folders to 100X in DMSO for convenience. For storage, seal tubes tightly.

PREPARATION OF WORKING SOLUTION

Add 20 µL of 500X ROS Brite™ 570 stock solution into 10 mL of Assay Buffer (Component B) and mix well to make ROS Brite™ 570 working solution. Note: This ROS Brite™ 570 working solution is stable for at least 2 hours at room temperature.

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

SAMPLE EXPERIMENTAL PROTOCOL

For Protocol A:

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

  2. To induce ROS, incubate the cell plate at room temperature or in a 5% CO2, 37 °C incubator for a desired period of time (for example: 30 minutes treatment for Hela cells with 100 µM tert-butyl hydroperoxide (TBHP)).

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of ROS Brite™ 570 working solution into the cell plate.

  4. Incubate the cells in a 5% CO2, 37 °C incubator for 30 min to 60 minutes.

  5. Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 540/570 nm (Cutoff = 550nm) or observe cells using a fluorescence microscope with TRITC filter set.

For Protocol B:

  1. Prepare cells at the density from 5 × 105 to 1 × 106 cells/mL. Note: Each cell line should be evaluated on the individual basis to determine the optimal cell density for apoptosis induction.

  2. Treat cells with test compounds in your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of compound buffer.

  3. To induce ROS, incubate the cell plate at room temperature or in a 5% CO2, 37 °C incubator for at least 30 minutes or a desired period of time (30 minutes for Hela cells treated with 100 µM tert-butyl hydroperoxide (TBHP)).

  4. Add 1 µL/mL cells of 500X ROS Brite™ 570 stock solution or 5 µL/mL cells of 100X ROS Brite™ 570 stock solution to cells medium.

  5. Incubate the cells in a 5% CO2, 37 °C incubator for 30 to 60 minutes.

  6. Monitor the fluorescence intensity using a flow cytometer with FL2 channel.

Citations


View all 10 citations: Citation Explorer
Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug design, development and therapy (2017): 3343
Notoginsenoside R1 attenuates high glucose-induced endothelial damage in rat retinal capillary endothelial cells by modulating the intracellular redox state
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug Design, Development and Therapy (2017): 3343
Anti-proliferation effect of blue light-emitting diodes against antibiotic-resistant Helicobacter pylori
Authors: Ma, Jianwei and Hiratsuka, Takahiro and Etoh, Tsuyoshi and Akada, Junko and Fujishima, Hajime and Shiraishi, Norio and Yamaoka, Yoshio and Inomata, Masafumi
Journal: Journal of Gastroenterology and Hepatology (2017)
Good hydration and cell-biological performances of superparamagnetic calcium phosphate cement with concentration-dependent osteogenesis and angiogenesis induced by ferric iron
Authors: Zhang, J and Shi, HS and Liu, JQ and Yu, T and Shen, ZH and Ye, JD
Journal: Journal of Materials Chemistry B (2015): 8782--8795
Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes
Authors: Patrick, Ping and Price, Tulin O and Diogo, Ana L and Sheibani, Nader and Banks, William A and Shah, Gul N
Journal: Journal of endocrinology and diabetes (2015)
Superoxide dismutase as a target of clioquinol-induced neurotoxicity
Authors: Kawamura, Kazuyuki and Kuroda, Yukiko and Sogo, Masako and Fujimoto, Miki and Inui, Toshio and Mitsui, Takao
Journal: Biochemical and biophysical research communications (2014): 181--185
Down-regulated peroxisome proliferator-activated receptor γ (PPARγ) in lung epithelial cells promotes a PPARγ agonist-reversible proinflammatory phenotype in chronic obstructive pulmonary disease (COPD)
Authors: Lakshmi, Sowmya P and Reddy, Aravind T and Zhang, Yingze and Sciurba, Frank C and Mallampalli, Rama K and Duncan, Steven R and Reddy, Raju C
Journal: Journal of Biological Chemistry (2014): 6383--6393
Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice
Authors: Nomura, Johji and Busso, Nathalie and Ives, Annette and Matsui, Chieko and Tsujimoto, Syunsuke and Shirakura, Takashi and Tamura, Mizuho and Kobayashi, Tsunefumi and So, Alex and er , undefined and Yamanaka, Yoshihiro
Journal: Scientific reports (2014): 4554
Oxidative Stress--An Update and Insight in the Romanian Family Physician’s Adoption of the Concept
Authors: Berghea, Florian and Berghea, Camelia Elena and Abobului, Mihai
Journal: Internal Medicine : 11--15

References


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Automatic flow injection based methodologies for determination of scavenging capacity against biologically relevant reactive species of oxygen and nitrogen
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Diabetes and the impairment of reproductive function: possible role of mitochondria and reactive oxygen species
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Journal: Curr Diabetes Rev (2008): 46
Virion disruption by ozone-mediated reactive oxygen species
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Journal: J Virol Methods (2008): 74
The role of mitochondria in reactive oxygen species metabolism and signaling
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Sensitive determination of reactive oxygen species by chemiluminescence methods and their application to biological samples and health foods
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Reactive oxygen species and yeast apoptosis
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Journal: Biochim Biophys Acta (2008): 1354
Measurement of reactive oxygen species in cells and mitochondria
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Journal: Methods Cell Biol (2007): 355
Role of reactive oxygen species in mediating hepatic ischemia-reperfusion injury and its therapeutic applications in liver transplantation
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Journal: Transplant Proc (2007): 1332
Superoxide and derived reactive oxygen species in the regulation of hypoxia-inducible factors
Authors: Gorlach A, Kietzmann T.
Journal: Methods Enzymol (2007): 421
Reactive oxygen species and superoxide dismutases: role in joint diseases
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