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

Detection of ROS in HeLa cells with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit. HeLa cells were seeded overnight at 15,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were untreated (control) or treated with 1 mM H<sub>2</sub>O<sub>2</sub> or 100 µM tert-butyl hydroperoxide (TBHP) for 30 minutes at 37 °C. The ROS Brite™ 670 working solution (100 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 1 hour. The fluorescence signal were monitored at Ex/Em = 650/675 nm (Cutoff = 665 nm) with bottom read mode using FlexStation (Molecular Devices).
Detection of ROS in HeLa cells with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit. HeLa cells were seeded overnight at 15,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were untreated (control) or treated with 1 mM H<sub>2</sub>O<sub>2</sub> or 100 µM tert-butyl hydroperoxide (TBHP) for 30 minutes at 37 °C. The ROS Brite™ 670 working solution (100 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 1 hour. The fluorescence signal were monitored at Ex/Em = 650/675 nm (Cutoff = 665 nm) with bottom read mode using FlexStation (Molecular Devices).
Images of Hela cells stained with the 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 30min before staining.
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™ 670 in a 5% CO<sub>2</sub>, 37 °C incubator for 1 hour. The fluorescence intensities were measured with APC channel using a flow cytometer (NovoCyte 3000, ACEA).
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Catalog Number22903
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Telephone1-408-733-1055
Fax1-408-733-1304
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H-phraseH303, H313, H333
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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™ 670 sensor to quantify ROS in live cells. The cell-permeable and non-fluorescent ROS Brite™ 670 exhibits a strong fluorescence signal upon reaction with ROS. ROS Brite™ 670 sensor is localized in the cytoplasm. The fluorescence signal of ROS Brite™ 670 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 Cy5 filter.

Platform


Flow cytometer

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC channel

Fluorescence microscope

ExcitationCy5 filter
EmissionCy5 filter
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation650 nm
Emission675 nm
Cutoff665 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: ROS Brite™ 6701 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™ 670 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= 650/675 nm (Cutoff = 665 nm) or fluorescence microscope with Cy5 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™ 670 with the cells for 30 - 60 minutes
  4. Monitor the fluorescence intensities using flow cytometer with APC channel 
Important      Thaw all the kit components at room temperature before starting the experiment.

CELL PREPARATION

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

PREPARATION OF STOCK SOLUTIONS

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.

ROS Brite™ 670 stock solution (500X)
Add 40 µL of DMSO (Component C) into the vial of ROS Brite™ 670 (Component A) and mix well to make 500X ROS Brite™ 670 stock solution. Protect from light.
Note      20 µL of 500X ROS Brite™ 670 stock solution is enough for 1 plate. For flow cytometer, 500X ROS Brite™ 670 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™ 670 stock solution into 10 mL of Assay Buffer (Component B) and mix well to make ROS Brite™ 670 working solution.
Note      This ROS Brite™ 670 working solution is stable for at least 2 hours at room temperature.

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™ 670 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 = 650/675 nm (Cutoff = 665 nm) or observe cells using a fluorescence microscope with Cy5 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™ 670 stock solution or 5 µL/mL cells of 100X ROS Brite™ 670 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 APC channel. 

Citations


View all 16 citations: Citation Explorer
Host Defense against Klebsiella pneumoniae Pneumonia Is Augmented by Lung-Derived Mesenchymal Stem Cells
Authors: Rangasamy, Tirumalai and Ghimire, Laxman and Jin, Liliang and Le, John and Periasamy, Sivakumar and Paudel, Sagar and Cai, Shanshan and Jeyaseelan, Samithamby
Journal: The Journal of Immunology (2021): 1112--1127
Low-power STED nanoscopy based on temporal and spatial modulation
Authors: Wang, Luwei and Chen, Yue and Guo, Yong and Xie, Weixin and Yang, Zhigang and Weng, Xiaoyu and Yan, Wei and Qu, Junle
Journal: Nano Research (2021): 1--8
Hypoxically cultured cells of oral squamous cell carcinoma increased their glucose metabolic activity under normoxic conditions
Authors: Shinohara, Yuta and Washio, Jumpei and Kobayashi, Yuri and Abiko, Yuki and Sasaki, Keiichi and Takahashi, Nobuhiro
Journal: Plos one (2021): e0254966
Tyrosine kinase inhibitor conjugated quantum dots for non-small cell lung cancer (NSCLC) treatment
Authors: Kulkarni, Nishant S and Parvathaneni, Vineela and Shukla, Snehal K and Barasa, Leonard and Perron, Jeanette C and Yoganathan, Sabesan and Muth, Aaron and Gupta, Vivek
Journal: European Journal of Pharmaceutical Sciences (2019)
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 (2018): 1492--1499
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)

References


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Diabetes and the impairment of reproductive function: possible role of mitochondria and reactive oxygen species
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Virion disruption by ozone-mediated reactive oxygen species
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The role of mitochondria in reactive oxygen species metabolism and signaling
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Measurement of reactive oxygen species in cells and mitochondria
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Superoxide and derived reactive oxygen species in the regulation of hypoxia-inducible factors
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