Iron colorimetric assay kit

Intracellular iron content was measured by Iron Colorimetric Assay Kit (Abcam, Cambridge, UK). To measure total iron, 5 µL Iron Reducer was added to each sample well to reduce Fe³⁺ to Fe²⁺, and incubated at 37 °C for 30 min. 100 µL Iron Probe was added to each well containing the Iron Standard and/or test samples. Total iron (Fe²⁺ + Fe³⁺) concentrations can be determined from the standard curve. The iron concentration was expressed as ng/µL.

Cells were cultured at 2 × 10⁵ cells/well in 6-well plate, ROS assay kit (Beyotime Biotech, Nanjing, China) was resorted to measuring generation of ROS by fluorescence microscopy and FCM. For lipid peroxidation, cells were incubated with 5 μM C11-BODIPY (D3861; Thermo Fisher, OR, USA), the signal were acquired by confocal laser scanning microscopy (CLSM, Leica TCS SP8; Leica Microsystems, Wetzlar, Germany) and FCM. Cells were plated in 6-well at 2 × 10⁵ cells/well, an iron colorimetric assay kit (Abcam, Cambridge, UK) was used to detect intracellular ferrous iron; absorbance at the wavelength of 593 nm was defined.

Various nodes during ferroptosis were analyzed using different methods. Firstly, intracellular Fe²⁺ was detected using the Iron Colorimetric Assay Kit (BioVision, Milpitas, CA, USA). In brief, cells were homogenized in 500 µL iron assay buffer and centrifuged at 16,000× g for 10 min. After incubating the plate for 30 min at 37 °C, a 100 µL iron probe was added to each well and incubated for 60 min at 37 °C. The absorbance at 593 nm was measured using a microplate reader against a standard curve. In addition, Fe²⁺ within the HEI-OC1 cells were further verified using the Intracellular Iron Measurement FerroOrange fluorescent probe that selectively binds iron ions (Dojindo Laboratories, Kumamoto, Japan). Secondly, the lipid peroxide within the cells was measured using the Liperfluo fluorescent probe, which reduces lipid hydroperoxide to their hydroxy homologues to yield a fluorescent product (Dojindo).

Heart tissue MDA levels, labile iron levels, and GSH levels were measured using commercial MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China), Iron Colorimetric Assay Kit (Cat#K390-100, BioVision, Milpitas, CA, USA), GSH-PX kit (Nanjing Jiancheng Bioengineering Institute, China), respectively, following the manufacturer's instructions [38 (link)].