Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Microplate Reader*
Overview | ![]() ![]() |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode |
Components
Component A: MitoROS™ 580 | 1 vial |
Component B: Assay Buffer | 1 bottle (20 mL) |
Component C: DMSO | 1 vial (100 µL) |
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells in growth medium
- Treat the cells with test compounds to induce superoxide
- Add MitoROS™ 580 working solution
- Stain the cells at 37°C for 30 - 60 minutes
- Monitor the fluorescence increase (bottom read mode) at Ex/Em= 540/590 nm (Cutoff = 570 nm) or fluorescence microscope with TRITC filter set
Important notes
Thaw all the components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. MitoROS™ 580 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ 580 (Component A) and mix well to make 500X MitoROS™ 580 stock solution. Protect from light. Note: 25 µL of 500X MitoROS™ 580 stock solution is enough for 1 plate. For storage, seal tubes tightly.
PREPARATION OF WORKING SOLUTION
Add 25 μL of 500X MitoROS™ 580 stock solution into 10 mL of Assay Buffer (Component B) and mix well to make MitoROS™ 580 working solution. Note: This MitoROS™ 580 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
- Treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X 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 superoxide, incubate the cell plate at 37°C for a desired period of time, protect from light. Note: We treated HeLa cells with 50 µM Antimycin A (AMA) at 37°C for 30 minutes to induce superoxide. See Figure 1 for details.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ 580 working solution into the cell plate.
- Incubate the cells at 37°C for 30 to 60 minutes.
- Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 m) or observe cells using a fluorescence microscope with TRITC filter.
Images
Citations
Authors: Cheng, Gang and Li, Lihuan
Journal: Journal of Biosciences (2020): 1--11
Authors: Pichla, Monika and Pulaski, {\L}ukasz and Kania, Katarzyna Dominika and Stefaniuk, Ireneusz and Cieniek, Bogumi{\l} and Pie{\'n}kowska, Natalia and Bartosz, Grzegorz and Sadowska-Bartosz, Izabela
Journal: Oxidative Medicine and Cellular Longevity (2020)
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Application notes
A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells
A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells
Activation of the Mitochondrial Apoptotic Pathway Produces Reactive Oxygen Species and Oxidative Damage in Hepatocytes That Contribute to Liver Tumorigenesis
Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma
Cold exposure lowers energy expenditure at the cellular level