Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit*Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells at a density of 0.5 - 1 × 10⁶ cells/mL
2. Treat the cells with test compounds to induce superoxide
3. Add 1 µL 500X MitoROS™ 580 into 0.5 mL cell suspension
4. Stain the cells at 37°C for 1 hour
5. Monitor the fluorescence intensity using flow cytometer with FL2 channel

Important notes
Thaw all the components at room temperature before starting the experiment.

1. MitoROS™ 580 stock solution (500X):
Add 100 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: For storage, seal tubes tightly.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. For each sample, prepare cells in 0.5 mL growth medium or buffer of your choice at a density of 5×10⁵ to 1×10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for superoxide induction.
   
   
2. Treat cells with 25 µL of 20X test compounds in Assay Buffer (Component B) or your desired buffer (such as PBS) to induce superoxide. For control cells (untreated cells), add the corresponding amount of compound buffer.
   
   
3. Incubate the cells at 37°C for at least 30 minutes or a desired period of time, protected from light. Note: Jurkat cells were treated with 50 µM Antimycin A (AMA) at 37°C for 30 minutes to induce superoxide. See Figure 1 for details. Pyocyanin (50 µM) or H₂O₂ (1 mM) can also be used to induce superoxide.
   
   
4. Add 1 µL of 500X MitoROS™ 580 stock solution into 0.5 mL cell suspension.
   
   
5. Incubate at 37°C for 1 hour. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with MitoROS™ 580. The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.
   
   
6. Monitor the fluorescence intensity using a flow cytometer with FL2 channel (Ex/Em=488/590 nm). Gate on the cells of interest, excluding debris.