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

Detection of ROS in Jurkat cells with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit. Jurkat cells were seeded on the same day at 300,000 cells/100µL/well in a Costar black wall/clear bottom 96-well plate. The ROS assay loading solution (100 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 1 hour. The cells were treated with or without 1 mM H<sub>2</sub>O<sub>2</sub> for 2 hours. The fluorescence signal was monitored at Ex/Em = 520/605 nm (Cutoff = 590 nm) with bottom read mode using FlexStation (Molecular Devices).
Detection of ROS in Jurkat cells with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit. Jurkat cells were seeded on the same day at 300,000 cells/100µL/well in a Costar black wall/clear bottom 96-well plate. The ROS assay loading solution (100 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 1 hour. The cells were treated with or without 1 mM H<sub>2</sub>O<sub>2</sub> for 2 hours. The fluorescence signal was monitored at Ex/Em = 520/605 nm (Cutoff = 590 nm) with bottom read mode using FlexStation (Molecular Devices).
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Catalog Number22901
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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
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OverviewpdfSDSpdfProtocol


Reactive oxygen species (ROS) are natural byproducts of the normal metabolism of oxygen and play important roles in cell signaling. However, ROS levels can increase dramatically during oxidative stress-related states, resulting in significant damage to cell structures. The role of oxidative stress in cardiovascular disease, diabetes, osteoporosis, stroke, inflammatory diseases, neurodegenerative disease, and cancer has been well established. Through ROS measurements, researchers can determine how oxidative stress modulates varied intracellular pathways. The Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit uses our unique ROS sensor, Amplite® ROS Red, to quantify ROS in live cells. Amplite® ROS Red is cell-permeable and generates red fluorescence when reacting with ROS. The Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay is easy to perform using an optimized mix-and-read format. It provides a sensitive, one-step fluorimetric assay to detect intracellular ROS in live cells with 1-2 hours incubation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using either a fluorescence microplate reader or a fluorescent microscope. It can be used to either quantify ROS activities or screen for ROS inhibitors.

Platform


Fluorescence microscope

Excitation520 nm
Emission605 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Texas Red filter set

Fluorescence microplate reader

Excitation520 nm
Emission605 nm
Cutoff590 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: Amplite™ ROS Red1 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: DMSO1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Add Amplite™ ROS Red working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
  3. Incubate the cells at 37°C for 1 hour
  4. Treat the cells with test compounds to induce ROS
  5. Monitor the fluorescence increase (bottom read mode) at Ex/Em= 520/605 nm (Cutoff = 590 nm) or fluorescence microscope with Ex/Em = 520/605 nm filter set

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. Amplite™ ROS Red stock solution (500X):
Add 40 µL of DMSO (Component C) into the vial of Amplite™ ROS Red (Component A) and mix well to make 500X Amplite™ ROS Red stock solution. Protect from light. Note: 20 µL of 500X Amplite™ ROS Red 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

Add 20 µL of 500X Amplite™ ROS Red stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Amplite™ ROS Red working solution. Note: This Amplite™ ROS Red 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

  1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Amplite™ ROS Red working solution into the cell plate.

  2. Incubate the cells in a 5% CO2, 37°C incubator for one hour.

  3. Treat cells with 20 µL of 11X test compounds (96-well plate) or 10 µL of 6X 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.

  4. To induce ROS, incubate the cell plate at room temperature or in a 5% CO2, 37°C incubator for at least 15 minutes or a desired period of time (30 minutes for Hela cells treated with 1 mM H2O2).

  5. Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 520/605 nm (Cutoff = 590 nm) or observe cells using a fluorescence microscope with Ex/Em = 520/605 nm filter set (Texas Red filter).

Citations


View all 21 citations: Citation Explorer
Suppression of optineurin impairs the progression of hepatocellular carcinoma through regulating mitophagy
Authors: Inokuchi, Shoichi and Yoshizumi, Tomoharu and Toshima, Takeo and Itoh, Shinji and Yugawa, Kyohei and Harada, Noboru and Mori, Hiroyuki and Fukuhara, Takasuke and Matsuura, Yoshiharu and Mori, Masaki
Journal: Cancer medicine (2021): 1501--1514
Human VAMP3 Suppresses or Negatively Regulates Bax Induced Apoptosis in Yeast
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Biomedicines (2021): 95
FK506-binding protein 2 (FKBP13) inhibit Bax-induced apoptosis in Saccharomyces cerevisiae (yeast)
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Cell Biology and Toxicology (2021): 1--10
Sensing the generation of intracellular free electrons using the inactive catalytic subunit of cytochrome P450s as a sink
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Sensors (2020): 4050
The effect of copy number on $\alpha$-synuclein’s toxicity and its protective role in Bax-induced apoptosis, in yeast
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Bioscience Reports (2020)
Identification of proteins involved in transcription/translation (eEF 1A1) as an inhibitor of Bax induced apoptosis
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Molecular biology reports (2020): 6785--6792
Nonantioxidant tetramethoxystilbene abrogates $\alpha$-synuclein-induced yeast cell death but not that triggered by the Bax or $\beta$A4 peptide
Authors: Derf, Asma and Mudududdla, Ramesh and Akintade, Damilare and Williams, Ibidapo S and Abdullaha, Mohd and Chaudhuri, Bhabatosh and Bharate, Sandip B
Journal: ACS omega (2018): 9513--9532
Inhibitors of Aβ42-induced endoplasmic reticular unfolded protein response (UPRER), in yeast, also rescue yeast cells from Aβ42-mediated apoptosis
Authors: Derf, Asma and Mudududdla, Ramesh and Bharate, S and ip B , undefined and Chaudhuri, Bhabatosh
Journal: European Journal of Pharmaceutical Sciences (2018)
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

References


View all 48 references: Citation Explorer
Automatic flow injection based methodologies for determination of scavenging capacity against biologically relevant reactive species of oxygen and nitrogen
Authors: Magalhaes LM, Lucio M, Segundo MA, Reis S, Lima JL.
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Diabetes and the impairment of reproductive function: possible role of mitochondria and reactive oxygen species
Authors: Amaral S, Oliveira PJ, Ramalho-Santos J.
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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Journal: Ann N Y Acad Sci (2008): 37
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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Superoxide and derived reactive oxygen species in the regulation of hypoxia-inducible factors
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Reactive oxygen species and superoxide dismutases: role in joint diseases
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