FerroOrange is a fluorescent probe designed to detect labile ferrous ions, localizing specifically to the endoplasmic reticulum. Upon interaction with ferrous ions, it irreversibly emits an orange fluorescence. The excitation/emission wavelengths are 542/572 nm.

To prepare the solution, add 35 µL of DMSO to a tube containing 24 µg of FerroOrange and mix thoroughly with a pipette to create a 1 mM FerroOrange solution. Dilute this 1 mM FerroOrange solution with HBSS to obtain a 1 µM FerroOrange working solution. Note that the 1 mM FerroOrange stock solution can be stored at -20℃ for up to one month, while the working solution is unstable and should be used immediately. If necessary, serum-free media may be used instead of HBSS to reduce background. For the procedure, culture cells in a fluorescence dish overnight at 37℃ with 5% CO2. Remove the supernatant and wash the cells three times with HBSS or serum-free medium. Replace with medium containing the drug and culture at 37℃ with 5% CO2, adjusting culture time as needed. Remove the medium and wash three times with HBSS or serum-free medium. Add the 1 µM FerroOrange working solution and incubate at 37℃ with 5% CO2 for 30 minutes. No washing is required; proceed directly to observation under a fluorescence microscope.