FerroBrite™ Green

Before initial use, thaw FerroBrite™ Green at room temperature and briefly centrifuge to collect the dried pellet.

1. Treat the cells as desired.

2. Remove the treatment and add 100 µL of FerroBrite™ Green working solution.

3. Incubate at 37 °C for 30 minutes.

4. Use an Ex/Em = 450/550 filter set to monitor the fluorescence signal.

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 FerroBrite™ Green stock solution in DMSO. For example, to make a 10 mM FerroBrite™ Green stock solution add 284 µL of DMSO to the vial of FerroBrite™ Green, and mix thoroughly.

   Note: Prepare single-use aliquots of any remaining FerroBrite™ Green stock solution. Store aliquots at ≤ -20 º C, protected from light. Avoid freeze/thaw cycles.

1. Prepare a 10 to 20 μM working solution by diluting the FerroBrite™ Green stock solution into Hanks' solution with 20 mM Hepes buffer (HHBS, AAT Cat No. 20011).

   Note: For optimal results, use this solution within 2 hours of preparation.

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

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

2. Add the drug of interest to the cells at the necessary concentration.

   Note: For a positive control, add Erastin at a concentration of 10 µM to the cells. Incubate the cells with Erastin for 5 to 6 hours at 37°C in a 5% CO2 incubator to induce ferroptosis.

3. Remove the cell culture medium and add 100 µL of FerroBrite™ Green working solution to the cells.

4. Incubate the cells at 37°C for 20 to 30 minutes, keeping them protected from light.

   Note: The optimal concentration and incubation time for FerroBrite™ Green may vary depending on the cell line used. It is recommended to test various concentrations to determine the most effective dose.

5. Remove the FerroBrite™ Green working solution and wash the cells twice using HHBS buffer.

6. Add HHBS buffer and analyze the cells using a fluorescence microscope with an Ex/Em = 450/550 nm filter set.