Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay KitDeep 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).

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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Telephone	1-800-990-8053
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Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

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

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

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

Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Optimized for Flow Cytometry*

Overview SDS Protocol

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

Excitation	640 nm laser
Emission	660/20 nm filter
Instrument specification(s)	APC channel

Fluorescence microscope

Excitation	Cy5 filter
Emission	Cy5 filter
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	650 nm
Emission	675 nm
Cutoff	665 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode

Components

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.

Images

Figure 1. 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 H₂O₂ 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).

Figure 2. 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.

Figure 3. 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% CO₂, 37 °C incubator for 1 hour. The fluorescence intensities were measured with APC channel using a flow cytometer (NovoCyte 3000, ACEA).

Citations

View all 22 citations: Citation Explorer

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

Bcl-2 interacting protein 3 (BNIP3) promotes tumor growth in breast cancer under hypoxic conditions through an autophagy-dependent pathway
Authors: Zhang, Guipu and Xu, Zhiyi and Yu, Minjing and Gao, Haiyan
Journal: Bioengineered (2022): 6280--6292

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

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)

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

Reactive oxygen species and yeast apoptosis
Authors: Perrone GG, Tan SX, Dawes IW.
Journal: Biochim Biophys Acta (2008): 1354

Measurement of reactive oxygen species in cells and mitochondria
Authors: Armstrong JS, Whiteman M.
Journal: Methods Cell Biol (2007): 355

Role of reactive oxygen species in mediating hepatic ischemia-reperfusion injury and its therapeutic applications in liver transplantation
Authors: Zhang W, Wang M, Xie HY, Zhou L, Meng XQ, Shi J, Zheng S.
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
Authors: Afonso V, Champy R, Mitrovic D, Collin P, Lomri A.
Journal: Joint Bone Spine (2007): 324