Ferroorange solution

A day prior to treatment, 6-well plates were used to seed cells in triplicate. They were pretreated with or without drugs. After 48 h, the cell culture medium in each well was replaced with fresh medium containing 1 µmol/L FerroOrange solution (Dojindo, China), and the incubation was carried out at 37 °C for 30 min. Afterward, cell suspensions were obtained, and the levels of Fe²⁺ were assessed using a flow cytometer.

The cells were trypsinized and transferred to confocal well in a volume of 100 μl with a cell density of 1 × 10⁴/ml. The cells were incubated with 1 μmol/L FerroOrange solution (Dojindo Molecular Technologies, Kumamoto, Japan) for 30 min and then observed under a confocal fluorescence microscope.
According to the manufacturer’s instructions, use the Iron Assay kit (Abcam) to determine the intracellular ferrous iron content. Collected 5 × 10⁶ cells to be tested were lysed with iron buffer, centrifuged at 12,000 rpm for 10 min to collect the supernatant, incubated with iron reducer reagent at 37°C for 30 min, and then incubated with iron probe at 37°C in dark for 1 h. The absorbance at 593 nm was measured with Microplate Reader.