Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Microplate Reader*

Protocol summary

1. Prepare cells in growth medium
2. Treat the cells with test compounds to induce superoxide
3. Add MitoROS™ 580 working solution
4. Stain the cells at 37°C for 30 - 60 minutes
5. 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.

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.

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

1. 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.
   
   
2. 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.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ 580 working solution into the cell plate.
   
   
4. Incubate the cells at 37°C for 30 to 60 minutes.
   
   
5. 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.