Key features and details
- Assay type: Cell-based
- Detection method: Fluorescent
- Platform: Microplate reader, Fluorescence microscope
- Sample type: Adherent cells, Suspension cells
Product nameMitochondrial Superoxide Assay Kit (Fluorometric)
Sample typeAdherent cells, Suspension cells
Species reactivityReacts with: Mammals, Other species
Mitochondrial Superoxide Detection Kit (Fluorometric) (ab219943) is a sensitive fluorometric one-step assay to detect intracellular superoxide radical in live cells. The assay uses our MitoROS 580 dye: the dye is cell-permeable and selectively reacts with mitochondrial superoxide present in live cells to generate a red fluorescence signal that can be easily read at Ex/Em = 540/590 nm.
The assay can be performed within one hour and can be detected by fluorescence microscopy, microplate reader or high-content imaging. It can be easily adapted to use in 384-well microplate format.
Mitochondria are major producers of cellular superoxide. The production of low to moderate levels of superoxide is critical for the proper regulation of many essential cellular processes including gene expression, signal transduction, and muscle adaptation to endurance exercise training. Uncontrolled mitochondrial superoxide production can trigger cellular oxidative damage that contributes to the pathogenesis of a wide variety of disorders including cancer, cardiovascular diseases, neurodegenerative diseases and aging. The detection of intracellular mitochondrial superoxide is of central importance to understanding proper cellular redox regulation and the impact of its dysregulation on various pathologies.
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.
PlatformMicroplate reader, Fluorescence microscope
Storage instructionsStore at -20°C. Please refer to protocols.
Components 200 tests Assay Buffer 1 x 20ml DMSO 1 x 100µl MitoROS™ 580 1 vial
Superoxide production in HeLa cells. HeLa cells were seeded overnight (105 cells/well/100 µL) in a 96 well black wall/clear bottom plate. Left: cells were treated with 50 µM Antimycin A (AMA) at 37ºC for 30 minutes, then incubated with MitoROS 580 for 1 hour. Right: control HeLa cells were incubated with MitoROS580 at 37 ºC for 1 hour without treatment. The fluorescence signal was measured using fluorescence microscope with a TRITC filter.
Quantification of superoxide production in HeLa cells. HeLa cells were seeded overnight (105 cells/well/100 µL) in a 96 well black wall/clear bottom plate. Cells were left untreated (control) or treated with either pyocyanin (Pyo, 50 µM Pyocyanin) or antimycin A (AMA, 50 µM Antimycin A) at 37 ºC for 30 minutes. Cells were then incubated with MitoROS 580 at 37 ºC for 1 hour. The fluorescence signal was monitored at Ex/Em = 540/590 nm (cut off = 570 nm) with bottom read mode using a microplate reader.
ab219943 has been referenced in 4 publications.
- Villa-Bellosta R Dietary magnesium supplementation improves lifespan in a mouse model of progeria. EMBO Mol Med 12:e12423 (2020). PubMed: 32875720
- Coates JTT et al. The anti-malarial drug atovaquone potentiates platinum-mediated cancer cell death by increasing oxidative stress. Cell Death Discov 6:110 (2020). PubMed: 33133645
- Patel SH et al. Advanced Glycation End-Products Suppress Mitochondrial Function and Proliferative Capacity of Achilles Tendon-Derived Fibroblasts. Sci Rep 9:12614 (2019). PubMed: 31471548
- Nguyen TT et al. Oxidative stress induced by Porphyromonas gingivalis lysate and nicotine in human periodontal ligament fibroblasts. Odontology N/A:N/A (2018). PubMed: 29959559