Cell Meter™ Fluorimetric Mitochondrial Superoxide Activity Assay Kit *Green Fluorescence*

Protocol summary (Fluorescence Microscope)

1. Prepare cells in growth medium
2. Treat the cells with test compounds to induce superoxide
3. Add MitoROS™ 520 working solution
4. Incubate the cells at 37°C for 1 hour
5. Monitor the fluorescence using FITC fliter set

Protocol summary (Flow Cytometry)

1. Prepare cells in growth medium
2. Treat the cells with test compounds to induce superoxide
3. Add MitoROS™ 520 stock solution and incubate the cells at 37°C for 1 hour
4. Monitor the fluorescence intensity with a flow cytometer using 530/30 nm filter (FITC channel)

Important notes
Thaw all the components at room temperature before use.

1. MitoROS™ 520 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ 520 (Component A) and mix well. Note: 25 µL of reconstituted MitoROS™ 520 stock solution is enough for 1 plate. Note: Unused portion can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.

Only for Fluorescence Microscope
Add 5 μL of 500X DMSO reconstituted MitoROS™ 520 stock solution into 2 mL of Assay Buffer (Component B) and mix well. Note: This working solution is not stable and needs to be prepared freshly before use.

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

For Fluorescence Microscopes/96-Well Microplates:

1. Treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in medium or 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 at 37°C for a desired period of time, protected from light. Note: We treated RAW 264.7 macrophage cells with 5 µM Antimycin A (AMA) at 37°C for 2 hours 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™ 520 working solution into the cell plate.
   
   
4. Incubate the cells at 37°C for 1 hour, and take images using fluorescence microscope with a FITC filter set.
   
   

For Flow Cytometers:

1. Treat cells as desired.
   
   
2. To induce superoxide, incubate the cells at 37°C for a desired period of time, protected from light. Note: We treated Jurkat cells with 50 µM Antimycin A (AMA) at 37°C for 2 hours to induce superoxide.
   
   
3. Add 1 µL/0.5 mL cells of MitoROS™ 520 stock solution (500X) into the cells.
   
   
4. Incubate the cells in a 5% CO₂, 37°C incubator for 1 hour, and monitor the fluorescence intensity using a flow cytometry with 530/30 nm filter (FITC channel).