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

Reactive oxygen species (ROS) are natural byproducts of the normal metabolism of oxygen and play important roles in cell signaling. However, during oxidative stress-related states, ROS levels can increase dramatically. 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 ROS Assay Kit uses our unique Amplite® ROS Green sensor to quantify ROS in live cells. Amplite® ROS Green is cell-permeable. It generates the green fluorescence when it reacts with ROS. The Cell Meter™ Fluorimetric ROS Assay Kit provides a sensitive, one-step fluorimetric assay to detect intracellular ROS in live cells with one hour incubation. This kit is optimized for flow cytometry applications, its signal can be detected with Ex/Em = 490/520 nm (FL1 channel).

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells at the density of 0.5 - 1 × 106 cells/mL
  2. Add 1 µL 500X Amplite™ ROS Green into 0.5 mL cell suspension
  3. Stain the cells at 37 ºC for 1 hour
  4. Treat the cells to induce ROS
  5. Analyze cells using flow cytometer with FL1 channel (Ex/Em = 490/520 nm)

Important notes
Thaw all the 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 Green stock solution (500X):
Add 100 µL of DMSO (Component C) into the vial of Amplite™ ROS Green (Component A) and mix well to make 500X Amplite™ ROS Green stock solution. Protect from light.  Note: For storage, seal tubes tightly.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 0.5 mL Assay Buffer (Component B) or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for ROS induction

  2. Add 1 µL of 500X Amplite™ ROS Green stock solution into 0.5 mL cell suspension.

  3. Incubate at 37 ºC for 1 hour. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Amplite™ ROS Green. The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.

  4. Treat cells by adding 50 µL of 11X test compounds in the desired buffer (such as PBS or HBSS). For control wells (untreated cells), add the corresponding amount of buffer. 

  5. Incubate the cells at 37 ºC for a desired period of time to induce ROS, protected from light. Note: We treated Jurkat cells with 100 µM TBHP (tert-Butyl hydroperoxide) at 37 ºC for 30 minutes to induce ROS. See Figure 1 for details.

  6. Monitor the fluorescence intensity using a flow cytometer with FL1 channel (Ex/Em = 490/520 nm). Gate on the cells of interest, excluding debris.

Citations

View all 20 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 December 6, 2024

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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Flow cytometer

Excitation488 nm laser
Emission530, 30 nm filter
Instrument specification(s)FITC channel

Components