Key features and details
- Assay type: Cell-based
- Platform: Flow cytometer, Fluorescence microscope
- Assay time: 90 min
- Sample type: Adherent cells, Suspension cells
Product nameROS/Superoxide Detection Assay Kit (Cell-based)
See all Oxidative Stress kits
Sample typeAdherent cells, Suspension cells
Assay time0h 90m
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
Reagents provided in the kit are sufficient for at least 200 tests using Fluorescent Microscopy or 50 tests using Flow Cytometry.
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.
To measure reactive oxygen species within cells, we recommend DCFDA / H2DCFDA - Cellular ROS Assay Kit ab113851. Alternative ROS assays are available in orange (ab186028), red (ab186027), and deep red (ab186029). ab238535 is used to measure ROS in biofluids, culture supernatants and cell lysates.
For assays designed to differentiate ROS, superoxides, and reactive nitrogen species: to assay ROS and superoxides use ab139476; to assay ROS, superoxides, and reactive nitrogen species use ab139473; to assay superoxides use ab219943.
PlatformFlow cytometer, Fluorescence microscope
Storage instructionsPlease refer to protocols.
Components 1 kit Oxidative Stress Detection Reagent (Green) 1 x 300nmole ROS Inducer (Pyocyanin 1 µmole) 1 vial ROS Inhibitor (N-acetyl-L-cysteine) 2 x 10mg Superoxide Detection Reagent (Orange) 1 x 300nmole Wash Buffer Salts 1 pack
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.
ab139476 has been referenced in 34 publications.
- Birinci Y et al. Quercetin in the form of a nano-antioxidant (QTiO2) provides stabilization of quercetin and maximizes its antioxidant capacity in the mouse fibroblast model. Enzyme Microb Technol 138:109559 (2020). PubMed: 32527528
- You L et al. Enterovirus 71 induces neural cell apoptosis and autophagy through promoting ACOX1 downregulation and ROS generation. Virulence 11:537-553 (2020). PubMed: 32434419
- Hu K et al. Marriage of black phosphorus and Cu2+ as effective photothermal agents for PET-guided combination cancer therapy. Nat Commun 11:2778 (2020). PubMed: 32513979
- Ren G et al. Reduced Basal Nitric Oxide Production Induces Precancerous Mammary Lesions via ERBB2 and TGFß. Sci Rep 9:6688 (2019). PubMed: 31040372
- Corsa CAS et al. The E3 ubiquitin ligase parkin is dispensable for metabolic homeostasis in murine pancreatic ß cells and adipocytes. J Biol Chem 294:7296-7307 (2019). PubMed: 30877201