Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Red Fluorescence*
Overview | ![]() ![]() |
Platform
Fluorescence microscope
Excitation | 520 nm |
Emission | 605 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Texas Red filter set |
Fluorescence microplate reader
Excitation | 520 nm |
Emission | 605 nm |
Cutoff | 590 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode |
Components
Component A: Amplite™ ROS Red | 1 vial |
Component B: Assay Buffer | 1 bottle (20 mL) |
Component C: DMSO | 1 vial (200 µL) |
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells in growth medium
- Add Amplite™ ROS Red working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
- Incubate the cells at 37°C for 1 hour
- Treat the cells with test compounds to induce ROS
- Monitor the fluorescence increase (bottom read mode) at Ex/Em= 520/605 nm (Cutoff = 590 nm) or fluorescence microscope with Ex/Em = 520/605 nm filter set
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Amplite™ ROS Red stock solution (500X):
Add 40 µL of DMSO (Component C) into the vial of Amplite™ ROS Red (Component A) and mix well to make 500X Amplite™ ROS Red stock solution. Protect from light. Note: 20 µL of 500X Amplite™ ROS Red stock solution is enough for 1 plate. Note: Unused portion can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.
PREPARATION OF WORKING SOLUTION
Add 20 µL of 500X Amplite™ ROS Red stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Amplite™ ROS Red working solution. Note: This Amplite™ ROS Red 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 Red 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 = 520/605 nm (Cutoff = 590 nm) or observe cells using a fluorescence microscope with Ex/Em = 520/605 nm filter set (Texas Red filter).
Images
Citations
Authors: Inokuchi, Shoichi and Yoshizumi, Tomoharu and Toshima, Takeo and Itoh, Shinji and Yugawa, Kyohei and Harada, Noboru and Mori, Hiroyuki and Fukuhara, Takasuke and Matsuura, Yoshiharu and Mori, Masaki
Journal: Cancer medicine (2021): 1501--1514
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Biomedicines (2021): 95
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Cell Biology and Toxicology (2021): 1--10
Authors: Akintade, DD and Chaudhuri, B
Journal: (2021)
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Molecular biology reports (2020): 6785--6792
Authors: Derf, Asma and Mudududdla, Ramesh and Akintade, Damilare and Williams, Ibidapo S and Abdullaha, Mohd and Chaudhuri, Bhabatosh and Bharate, Sandip B
Journal: ACS omega (2018): 9513--9532
Authors: Derf, Asma and Mudududdla, Ramesh and Bharate, S and ip B , undefined and Chaudhuri, Bhabatosh
Journal: European Journal of Pharmaceutical Sciences (2018)
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug design, development and therapy (2017): 3343
Authors: Fan, Chunlan and Qiao, Yuan and Tang, Minke
Journal: Drug Design, Development and Therapy (2017): 3343
Authors: Russo, Annapina and Saide, Assunta and Smaldone, Silvia and Faraonio, Raffaella and Russo, Giulia
Journal: International journal of molecular sciences (2017): 547
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Application notes
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