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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.
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/90&micro;L/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 &micro;M tert-butyl hydroperoxide (TBHP) for 30min at 37 &deg;C. The ROS Brite&trade; 570 assay solution (100&micro;L/well) was added and incubated in a 5% CO<sub>2</sub>, 37 &deg;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 100&micro;M tert-butyl hydroperoxide (TBHP) (Red) for 30min at 37 &deg;C, and then loaded with ROS Brite&trade; 570 in a 5% CO<sub>2</sub>, 37 &deg;C incubator for 1 hour. The fluorescence intensities were measured with Acea flow cytometer using&nbsp;PE channel.
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H-phraseH303, H313, H333
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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. 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


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 Note

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™ 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.

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 solution.

  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.

Images


Citations


View all 17 citations: Citation Explorer
Linoleic acid exhibits anti-proliferative and anti-invasive activities in endometrial cancer cells and a transgenic model of endometrial cancer
Authors: Qiu, Jianqing and Zhao, Ziyi and Suo, Hongyan and Paraghamian, Sarah E and Hawkins, Gabrielle M and Sun, Wenchuan and Zhang, Xin and Hao, Tianran and Deng, Beor and Shen, Xiaochang and others,
Journal: Cancer Biology \& Therapy (2024): 2325130
Reduced expression of phosphorylated ataxia-telangiectasia mutated gene is related to poor prognosis and gemcitabine chemoresistance in pancreatic cancer
Authors: Xun, Jingyu and Ohtsuka, Hideo and Hirose, Katsuya and Douchi, Daisuke and Nakayama, Shun and Ishida, Masaharu and Miura, Takayuki and Ariake, Kyohei and Mizuma, Masamichi and Nakagawa, Kei and others,
Journal: BMC Cancer (2023): 1--13
Anti-Inflammatory Effects of $\beta$-Cryptoxanthin on 5-Fluorouracil-Induced Cytokine Expression in Human Oral Mucosal Keratinocytes
Authors: Yamanobe, Hironaka and Yamamoto, Kenta and Kishimoto, Saki and Nakai, Kei and Oseko, Fumishige and Yamamoto, Toshiro and Mazda, Osam and Kanamura, Narisato
Journal: Molecules (2023): 2935
Obstructive sleep apnea-increased DEC1 regulates systemic inflammation and oxidative stress that promotes development of pulmonary arterial hypertension
Authors: Li, Xiaoming and Zhang, Xiang and Hou, Xiaozhi and Bing, Xin and Zhu, Fangyuan and Wu, Xinhao and Guo, Na and Zhao, Hui and Xu, Fenglei and Xia, Ming
Journal: Apoptosis (2022): 1--15
ONC206 has anti-tumorigenic effects in human ovarian cancer cells and in a transgenic mouse model of high-grade serous ovarian cancer
Authors: Tucker, Katherine and Yin, Yajie and Staley, Stuart-Allison and Zhao, Ziyi and Fang, Ziwei and Fan, Yali and Zhang, Xin and Suo, Hongyan and Sun, Wenchuan and Prabhu, Varun Vijay and others,
Journal: American Journal of Cancer Research (2022): 521
Reversal of multidrug resistance by Fissistigma latifolium--derived chalconoid 2-hydroxy-4, 5, 6-trimethoxydihydrochalcone in cancer cell lines overexpressing human P-glycoprotein
Authors: Teng, Yu-Ning and Hung, Chin-Chuan and Kao, Pei-Heng and Chang, Ying-Tzu and Lan, Yu-Hsuan
Journal: Biomedicine \& Pharmacotherapy (2022): 113832
The Abnormal Proliferation of Hepatocytes is Associated with MC-LR and C-Terminal Truncated HBX Synergistic Disturbance of the Redox Balance
Authors: Cai, Dong-Mei and Mei, Fan-Biao and Zhang, Chao-Jun and An, San-Chun and Lv, Rui-Bo and Ren, Guan-Hua and Xiao, Chan-Chan and Long, Long and Huang, Tian-Ren and Deng, Wei
Journal: Journal of Hepatocellular Carcinoma (2022): 1229--1246
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)

References


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