Ferroorange

Serum and retinal iron levels (total iron, ferrous iron, and ferric iron) were measured using an Iron Assay Kit (MAK025; Sigma) according to the manufacturer’s instructions. Perl’s staining was used to detect the ferric iron distribution in each layer of the retina. Briefly, deparaffinized and rehydrated retinal paraffin sections (3 µm) were stained using a Prussian blue staining kit (G1029; Servicebio) according to the recommended protocol. FerroOrange (F374; Dojindo, Beijing, China) was applied to reveal the distribution and expression of ferrous iron in the cytoplasm of living R28 cells. Briefly, the R28 cell culture medium was replaced with serum-free medium containing 1 μM FerroOrange, and then the R28 cells were incubated at 37 °C with an atmosphere containing 5% CO₂ for 30 min before being detected using a confocal fluorescence microscope (Leica, Frankfurt, Germany).

FerroOrange (F374, Dojindo Molecular Technologies Inc) was used to detect the cellular iron. Briefly, HCT116 ‐MOCK and ‐CYGB cells were seeded on microscope cover glass in culture dishes for 24 hours and stained with FerroOrange (0.5 μM) for 30 minutes, washed and then observed under an inverted confocal laser scanning microscope equipped with a 60 × oil immersion optic (Leica, TCS SP8).