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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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InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


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.


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


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

Example protocol


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.


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.


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


  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.


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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,
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Protection by Collagen Peptides from Walleye Pollock Skin on Bone Formation via Inhibition of Oxidative Stress
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Activation of p53-Mediated Apoptosis Pathway in HSC3 Cancer Cell Irradiated by Atmospheric DBD Oxygen Plasma
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