Ferroorange working solution

Cells in different treatment groups were washed once with PBS. Cells were then stained with FerroOrange working solution (DOJINDO) at a concentration of 1 μM and incubated at 37°C for 30 min. Flow cytometry or an Operetta High-Content Screening System of a confocal microscope (PerkinElmer) was used to detect at a wavelength of Ex: 561 nm/Em: 570–620 nm.

MDA-MB-231 cells were inoculated into eight-chambered slides and cultivated until the fusion rate reached 60-70%. Next, the original medium was discarded and replaced with fresh medium containing the drugs at the indicated concentrations. After incubation at 37°C for 24 h, the cells were washed with serum-free medium thrice. Subsequently, 1 µmol/L FerroOrange working solution (F374; Dojindo Laboratories Inc.) was added into each chamber and cultivated in 5% CO₂ at 37°C for 30 min. Thereafter, cell samples were imaged using a real-time live-cell laser scanning confocal microscope (UltraVIEW VOX; PerkinElmer, Inc.). The relative mean fluorescence intensity (MFI) of intracellular ferrous ions in different microscopic fields was calculated and analyzed using ImageJ software.

C-33A and HeLa cells were seeded into 8-well plates and incubated overnight. The cells were then replenished with fresh medium containing drugs at indicated concentrations and incubated for 24 h. Then, the cells were washed with serum-free medium 3 times; 1 μmol/l of FerroOrange working solution (Dojindo Laboratories, Japan) was added into each well and incubated for 30 min. The cells were imaged with a laser scanning confocal microscope (UltraVIEW VOX, PerkinElmer, Inc., Waltham, MA, United States). Relative mean fluorescence intensity (MFI) of ferrous ions was calculated using ImageJ software.