Ab83366

The concentration of ferrous iron (Fe²⁺) was measured using an iron colorimetric assay kit (ab83366, Abcam, USA) according to the manufacturer’s instructions. After CXCL2 or control plasmids overexpression, Huh7 and MHCC97H cells were treated with erastin (10 μM) for 24 hours. Cells were harvested using trypsin without EDTA and homogenized in iron assay buffer on ice, then centrifuged at 4°C (14,000×g, 15 min) to remove insoluble material. Subsequently, collect the supernatant and add assay buffer, mix and incubate for 30 min at 25°C. Add 100ul iron probe into each sample and incubate at 25°C for 60 min protected from light. Detect the absorbance at 593 nm using the VICTOR X2 microplate reader (PerkinElmer, Waltham, USA).

Commercially available enzyme-linked immunosorbant assay (ELISA) kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), amylase (BioVision, K711), MPO (BioVision, K744), iron (Abcam, ab83366), MDA (Abcam, ab118970), and 8-OHdG (Cell Biolabs, STA-320) in indicated samples according to the manufacturer’s instructions. Data were normalized to protein or DNA concentration. In addition, C11-BODIPY probe (Thermo Fisher Scientific, D3861) was used to detect lipid ROS in cells.

The concentration of ferrous (Fe²⁺) and/or ferric (Fe³⁺) iron in biological samples could be determined using an iron assay kit (Abcam, ab83366). In brief, in the acid assay buffer, the ferric carrier proteins could dissociate ferric into the solution. Then, the reaction of free ferrous iron with Ferene S results in stable-colored complexes with absorbance at 593 nm. Therefore, the levels of intracellular iron can be identified by measuring the absorbance at 593 nm.