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

Fluorescence images of simultaneous detection of intracellular nitric oxide (NO) and total ROS in RAW 264.7 macrophage. Cells were co-stained with Nitrixyte™ Orange (Red) and Amplite™ ROS Green (Green). The cells were then treated with or without 20 µg/mL of lipopolysaccharide (LPS), 1 mM L-arginine (L-Arg) and 50 µM Pyocyanin (Pyo) at 37°C for 16 hours. The fluorescence signals were measured using fluorescence microscope equipped with TRITC (Nitrixyte™ Orange, Red) and FITC (Amplite™ ROS Green, Green) filter sets, simultaneously.
Fluorescence images of simultaneous detection of intracellular nitric oxide (NO) and total ROS in RAW 264.7 macrophage. Cells were co-stained with Nitrixyte™ Orange (Red) and Amplite™ ROS Green (Green). The cells were then treated with or without 20 µg/mL of lipopolysaccharide (LPS), 1 mM L-arginine (L-Arg) and 50 µM Pyocyanin (Pyo) at 37°C for 16 hours. The fluorescence signals were measured using fluorescence microscope equipped with TRITC (Nitrixyte™ Orange, Red) and FITC (Amplite™ ROS Green, Green) filter sets, simultaneously.
Detection of ROS in Jurkat cells with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit. Jurkat cells were seeded on the same day at 300,000 cells/100µL/well in a Costar black wall/clear bottom 96-well plate. The ROS assay loading solution (100 µL/well) was added and incubated in a 5% CO2, 37 °C incubator for 1 hour. And then the cells were treated with 1mM, 0.1mM H<sub>2</sub>O<sub>2</sub> or without H<sub>2</sub>O<sub>2</sub> for 30 minutes. The fluorescence signal was monitored at Ex/Em = 490/525 nm (cutoff at 515 nm) with bottom read mode using FlexStation (Molecular Devices).
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Catalog Number22900
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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H-phraseH303, H313, H333
Hazard symbolXN
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. 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 ROS sensor to quantify ROS in live cells. ROS Green is cell-permeable. It generates the green fluorescence when it reacts with ROS. The kit is an optimized "mix and read" assay format that is compatible with HTS liquid handling instruments. 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. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using either a fluorescence microplate reader or a fluorescence microscope.

Platform


Fluorescence microscope

ExcitationFITC filter
EmissionFITC filter
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: Amplite™ ROS Green1 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: DMSO1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium
  2. Add Amplite™ ROS Green working solution (100 µL/well for a 96- well plate or 25 µL/well for a 384-well plate) 
  3. Stain the cells at 37°C for 60 minutes
  4. Treat the cells with test compounds to induce ROS
  5. Monitor the fluorescence increase (bottom read mode) at Ex/Em= 490/525 nm (Cutoff = 515 nm) or fluorescence microscope with FITC filter set

Important notes
Thaw all the kit 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 40 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: 20 µL of 500X Amplite™ ROS Green stock solution is enough for 1 plate. For storage, seal tubes tightly.

PREPARATION OF WORKING SOLUTION

Add 20 µL of 500X Amplite™ ROS Green stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Amplite™ ROS Green working solution. Note: This Amplite™ ROS Green working solution is stable for at least 2 hours at room temperature.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Amplite™ ROS Green working solution into the cell plate.

  2. Incubate the cells in a 5% CO2, 37°C incubator for one hour.

  3. Treat cells with 20 µL of 11X test compounds (96-well plate) or 10 µL of 6X 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.

  4. To induce ROS, incubate the cell plate at room temperature or in a 5% CO2, 37°C incubator for at least 15 minutes or a desired period of time (30 minutes for Hela cells treated with 1 mM H2O2).

  5. Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) or observe cells using a fluorescence microscope with FITC filter set.

Citations


View all 23 citations: Citation Explorer
Antineoplastic Effects and Mechanisms of a New RGD Chimeric Peptide from Bullfrog Skin on the Proliferation and Apoptosis of B16F10 Cells
Authors: Jiang, Xuan and Zhang, Xin and Fu, Chao and Zhao, Ruili and Jin, Tianming and Liu, Mengyue and Pan, Chenhao and Li, Liu An and Ma, Jifei and Yu, Enyuan and others,
Journal: The Protein Journal (2021): 1--12
ALS-causing SOD1 mutants regulate occludin phosphorylation/ubiquitination and endocytic trafficking via the ITCH/Eps15/Rab5 axis
Authors: Tang, Jingshu and Kang, Yuying and Zhou, Yujun and Li, Xinnan and Lan, Jiaqi and Wu, Lei and Feng, Xinhong and Peng, Ying
Journal: Neurobiology of Disease (2021): 105315
Hydrogen peroxide induces progranulin expression to control neurite outgrowth in HT22 cells
Authors: Shimoda, Ayumu and Tanabe, Takemi and Sato, Tsubasa and Nedachi, Taku
Journal: Bioscience, Biotechnology, and Biochemistry (2021): 2103--2112
Caffeic Acid Attenuates Multi-Drug Resistance in Cancer Cells by Inhibiting Efflux Function of Human P-glycoprotein
Authors: Teng, Yu-Ning and Wang, Charles CN and Liao, Wei-Chieh and Lan, Yu-Hsuan and Hung, Chin-Chuan
Journal: Molecules (2020): 247
Epigallocatechin gallate diminishes cigarette smoke-induced oxidative stress, lipid peroxidation, and inflammation in human bronchial epithelial cells
Authors: Lakshmi, Sowmya P and Reddy, Aravind T and Kodidhela, Lakshmi Devi and Varadacharyulu, N Ch
Journal: Life Sciences (2020): 118260
Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness
Authors: Bayne, Mitchell and Alvarsson, Alexandra and Devarakonda, Kavya and Li, Rosemary and Jimenez-Gonzalez, Maria and Garibay, Darline and Conner, Kaetlyn and Varghese, Merina and Serasinghe, Madhavika N and Chipuk, Jerry E and others,
Journal: JCI insight (2020)
Protection by Collagen Peptides from Walleye Pollock Skin on Bone Formation via Inhibition of Oxidative Stress
Authors: Liu, Feng and Wang, Wencheng and Xia, Yunqiu and Chen, Xuehong and Han, Yantao and Miao, Desen and Zhang, Deyan and Lv, Hong and Yang, Desheng and Zhang, Daisong and others,
Journal: Journal of King Saud University-Science (2020)
Organogermanium suppresses cell death due to oxidative stress in normal human dermal fibroblasts
Authors: Takeda, Tomoya and Doiyama, Sota and Azumi, Junya and Shimada, Yasuhiro and Tokuji, Yoshihiko and Yamaguchi, Hiroaki and Nagata, Kosuke and Sakamoto, Naoya and Aso, Hisashi and Nakamura, Takashi
Journal: Scientific reports (2019): 1--17
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)
Activation of p53-Mediated Apoptosis Pathway in HSC3 Cancer Cell Irradiated by Atmospheric DBD Oxygen Plasma
Authors: Hayashi, Nobuya and Miyamaru, Yukie and Aijima, Reona and Yamashita, Yoshio
Journal: IEEE Transactions on Plasma Science (2018): 1--7

References


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