Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Green Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Platform
Fluorescence microscope
Excitation | FITC filter |
Emission | FITC filter |
Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 525 nm |
Cutoff | 515 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells in growth medium
- Add Amplite™ ROS Green 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 60 minutes
- Treat the cells with test compounds to induce ROS
- 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
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
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Amplite™ ROS Green working solution into the cell plate.
- Incubate the cells in a 5% CO2, 37°C incubator for one hour.
- 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.
- 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).
- 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.
Images
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-fluorimetric-intracellular-total-ros-activity-assay-kit-green-fluorescence%2Ffigure-for-cell-meter-fluorimetric-intracellular-total-ros-activity-assay-kit-green-fluorescence_UKG1X.jpg&w=3840&q=75)
![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).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-fluorimetric-intracellular-total-ros-activity-assay-kit-green-fluorescence%2Ffigure-for-cell-meter-fluorimetric-intracellular-total-ros-activity-assay-kit-green-fluorescence_b98f5.jpg&w=3840&q=75)
Citations
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
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
Authors: Zhang, Hongchen and Feng, Yuan and Si, Yanfang and Lu, Chuanhao and Wang, Juan and Wang, Shiquan and Li, Liang and Xie, Wenyu and Yue, Zheming and Yong, Jia and others,
Journal: Redox Biology (2023): 102983
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
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
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
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
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
Authors: Li, Jing and Qi, Fangzheng and Su, Huishan and Zhang, Chuanshan and Zhang, Qing and Chen, Ying and Chen, Ping and Su, Linjia and Chen, Yanan and Yang, Yuqi and others,
Journal: International journal of biological sciences (2022): 2914
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
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Application notes
Abbreviation of Common Chemical Compounds Related to Peptides
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity
Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function
Dopamine-Mediated Oxidation of Methionine 127 in α-Synuclein