Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay KitDeep Red Fluorescence

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.

Example protocol

AT A GLANCE

Protocol A summary (Fluorescence microplate reader, fluorescence microscope)

Prepare cells in growth medium
Treat the cells with test compounds to induce ROS
Add ROS Brite™ 670 working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
Stain the cells at 37 °C for 30 - 60 minutes
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)

Prepare cells in growth medium
Treat cells with test compounds to induce ROS
Incubate ROS Brite™ 670 with the cells for 30 - 60 minutes
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:

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.
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)).
Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of ROS Brite™ 670 working solution into the cell plate.
Incubate the cells in a 5% CO₂, 37 °C incubator for 30 min to 60 minutes.
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:

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.
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.
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)).
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.
Incubate the cells in a 5% CO₂, 37 °C incubator for 30 to 60 minutes.
Monitor the fluorescence intensity using a flow cytometer with APC channel.

Citations

View all 25 citations: Citation Explorer

Palmitic Acid Exerts Anti-Tumorigenic Activities by Modulating Cellular Stress and Lipid Droplet Formation in Endometrial Cancer
Authors: Zhao, Ziyi and Wang, Jiandong and Kong, Weimin and Newton, Meredith A and Burkett, Wesley C and Sun, Wenchuan and Buckingham, Lindsey and O’Donnell, Jillian and Suo, Hongyan and Deng, Boer and others,
Journal: Biomolecules (2024): 601

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

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.
Journal: Talanta (2009): 1219

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
Authors: Murray BK, Ohmine S, Tomer DP, Jensen KJ, Johnson FB, Kirsi JJ, Robison RA, O'Neill KL.
Journal: J Virol Methods (2008): 74

The role of mitochondria in reactive oxygen species metabolism and signaling
Authors: Starkov AA., undefined
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
Authors: Wada M., undefined
Journal: Yakugaku Zasshi (2008): 1031

Page updated on November 21, 2024

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