MitoROS Brite™ 670 *Optimized for Detecting Reactive Oxygen Species (ROS) in Mitochondria*

Before use, thaw MitoROS Brite™ 670 at room temperature. Once thawed, briefly centrifuge to collect the dried pellet.

1. Prepare the cells in a growth medium.

2. Stain the cells using the MitoROS Brite™ 670 working solution.

3. Treat the cells with your desired test compounds.

4. Monitor fluorescence intensity with a Cy5 filter set or Ex/Em = 650/670 nm.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Prepare a 5 to 10 mM MitoROS Brite™ 670 stock solution in DMSO.

   Note: Prepare single-use aliquots of the MitoROS Brite™ 670 stock solution and store at ≤ -20°C, protected from light. Avoid freeze-thaw cycles.

1. Prepare a 5 to 10 μM MitoROS Brite™ 670 working solution by diluting the MitoROS Brite™ 670 stock solution into Hanks solution with 20 mM Hepes buffer (HHBS).

   Note: For optimal results, use this solution within a few hours of preparation.

   Note: Protect the working solution from light by covering it with foil or storing it in a dark place.

1. Plate the cells as desired in a 96-well black wall-clear bottom plate.

2. Add 100 µL of the MitoROS Brite™ 670 working solution to the cells.

3. Incubate the cells at 37°C for 30 to 60 minutes, protected from light.

   Note: The optimal concentration and incubation time for MitoROS Brite™ 670 may vary between different cell lines. You may need to test different concentrations to determine the best conditions.

4. Remove the dye working solution and wash the cells twice with HHBS buffer.

5. Treat cells as desired.

6. Remove the treatment and wash cells twice with HHBS buffer.

7. Add HHBS buffer and examine the cells using a fluorescence microscope with a Cy5 filter set.