• Product name

    ROS/Superoxide Detection Assay Kit (Cell-based)
    See all Oxidative Stress kits
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

  • Assay time

    0h 90m
  • Product overview

    ROS/Superoxide Detection Assay Kit ab139476 is designed to directly monitor real time reactive oxygen species (ROS) production in live cells using fluorescence microscopy, flow cytometry, or microplate reader.

    The ROS/superoxide assay protocol is based on two fluorescent dyes: Oxidative Stress Detection Reagent (Green, Ex/Em 490/525 nm) for the detection of total ROS, and Superoxide Detection Reagent (Orange, Ex/Em 550/620 nm).

    Through the combination of these two specific fluorescent probes, the kit provides a simple and specific assay for the real-time measurement of global levels of total reactive oxygen species (ROS), and of superoxides, in living cells. The kit is compatible with the major components of tissue culture media (phenol red, FBS and BSA).

    ROS/superoxide assay protocol summary:
    - add ROS inhibitor to negative control cells and incubate for 30 min
    - add ROS/superoxide detection mix (with the addition of ROS inducer for positive control cells) and incubate for 30-60 min
    - remove and discard the detection mix liquid
    - wash samples and analyze with fluorescence microscope, or analyze by flow cytometry or microplate reader without washing

  • Notes

    Reagents provided in the kit are sufficient for at least 200 tests using Fluorescent Microscopy or 50 tests using Flow Cytometry.

    Related products

    Review the oxidative stress marker and assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Platform

    Flow cytometer, Fluorescence microscope



  • The level of ROS was analyzed in untreated compared to 2250 treated cells, additional NAC treatment served as a neg. Control
  • Profiling of reactive oxygen species formation by Fluorescence Microscopy was achieved in HeLa cells loaded with ROS/Superoxide detection reagents (ab139476) and treated with pyocyanin. General oxidative stress levels were monitored in the green channel, while superoxide production was detected in the orange channel. Pretreatment with NAC, a general ROS inhibitor, prevents formation of ROS.
  • Jurkat cells were induced with 100 µM pyocyanin (general ROS inducer, panel B), 200 µM antimycin A (superoxide inducer, panel C) or 1 µM of t-butylhydroperoxide (peroxide inducer, panel D), stained with two color ROS Detection Kit and analyzed using flow cytometry. Untreated cells (panel A) were used as a control. Cell debris were ungated and compensation was performed using single stained pyocyanin-treated samples. Red numbers reflect the percentage of the cells in each quadrant.



This product has been referenced in:

  • De Grandis RA  et al. Novel lawsone-containing ruthenium(II) complexes: Synthesis, characterization and anticancer activity on 2D and 3D spheroid models of prostate cancer cells. Bioorg Chem 85:455-468 (2019). Read more (PubMed: 30776556) »
  • Ramella M  et al. Effect of Cyclic Stretch on Vascular Endothelial Cells and Abdominal Aortic Aneurysm (AAA): Role in the Inflammatory Response. Int J Mol Sci 20:N/A (2019). Read more (PubMed: 30642067) »
See all 18 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A


We have not yet tested ab139476 Cellular ROS/Superoxide Detection Assay in kit in bacteria cells or e.coli cells. 

Our lab specialists informed me that the dyes should be actually permeable to the bacterial cell wall and therefore enter the bacteria cells. However, guidance on loading time will likely need to be changed and optimized individually by the end user.  Since we have never performed such experiment, we cannot provide any further suggestions or recommendation about protocol optimization. Another point to consider is that the lipophilic dyes cross the plasma membrane (in this case the cell wall) in an inactive state. Once inside the cell they are activated by esterases. As far as we know, bacteria have esterases so there should not be a problem.

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After reconstitution the dye is not extremely stable and will lose effectiveness if stored for longer than one week.

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The lab says fixation will cause the dyes to leak from the cells. I think the assumption is that fixation may damage cell membranes. I did find a paper which describes fixation using methanol or acetic acid, after staining with a DCFDA derivative. DCFDA is the reagent that stains ROS in the kit ab113851, and I suspect that it is the green dye in the other two kits, the ROS/NOS and superoxide assays, though our source for those kits has not confirmed. A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses. Shen et al, 2013. PMID: 23178299 http://www.ncbi.nlm.nih.gov/pubmed/23178299

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