Ferroorange assay

Labile cellular iron (LCI) is an intracellular transient redox-active iron that can induce the production of ROS via the Fenton reaction. The level of LCI was measured via FerroOrange assay (Dojindo, Japan). Briefly, The Huh7 cells were seeded in 96-well culture plates at a density of 1 × 10⁴ cells/well and pretreated with various concentrations of ginger extract (0–12.5 µg/mL) and DFP (100 µM) at 37 °C for 24 h. Then, the cells were treated with 0.2 mM FAS for 1 h to load iron into the cells. Afterward, iron-loaded cells were washed twice with Hank’s Balanced Salt Solution (HBSS) to remove the extracellular iron. Finally, 1 mM FerroOrange was added and incubated in the dark at 37 °C for 30 min. The fluorescence intensity was monitored at the excitation wavelength of 556 nm and the emission wavelength of 615 nm.

Intracellular total iron and ferrous iron concentrations were detected by an iron assay kit following the manufacturer’s protocol (Sigma-Aldrich, USA). A FerroOrange assay (Dojindo, Japan) was used to detect the ferrous iron level, and the mitochondrial membrane potential was detected by a Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, China). The TEM samples were prepared using our previously described method, and ultra-structural images were captured with the transmission electron micorscope (JEM-1011, Japan) (18 (link)).

For quantifying intracellular iron levels, the Iron Assay Kit (DOJINDO, I291, Japan) was employed. After the treatments, mouse kidney tissue and HK-2 cell samples were lysed using the iron detection buffer provided. Following the guidelines provided by the kit, the absorbance of the reaction mix was assessed at 593 nm with a microplate reader. Furthermore, the FerroOrange assay (DOJINDO, F374, Japan) facilitated the visual assessment of intracellular Fe²⁺ levels. The presence of Fe²⁺ within the cells was detected using a 400× magnification inverted fluorescence microscope (Olympus, IX71, Japan).