E bc k881 m

Alterations in iron levels serve as a pivotal biomarker for identifying ferroptosis. Intracellular total iron and ferrous ion content were measured using the iron assay kits (E‐BC‐K880‐M and E‐BC‐K881‐M, Elabscience), respectively. Briefly, cells were collected with a cell scraper and 0.2 mL of buffer lysate was added to approximately 1 × 10⁶ cells per sample. The mixture was evenly mixed, incubated on ice for 10 min, centrifuged at 15000 × g for 10 min, and the supernatant was used for measurement following kit instructions. Total iron samples were incubated at 37°C for 40 min, while ferrous content samples were incubated at 37°C for 10 min before measuring absorbance at 593 nm using an enzyme‐labelled. Calculate the total iron and ferrous content according to the instructions.

A cell ferrous iron colorimetric assay (E-BC-K881-M, Elabscience Biotechnology Inc., Houston, TX, USA) was performed to measure the intracellular Fe²⁺ content, following the manufacturer’s instructions. After transfection, 1 × 10⁶ cells were centrifuged for 10 min at 1500× g at 4 °C and then incubated with 80 μL of reagent for 10 min at 37 °C. The 593 nm absorbance was measured with the microplate reader (model 680, Bio-Rad, Hercules, CA, USA), and the Fe²⁺ concentration was derived from the cell ferrous iron standard curve. Ferroptosis was investigated by using the fluorescent probe FerroOrange (F374, Dojindo Molecular Technologies, Inc., Rockville, MD, USA), according to the supplier’s guidance. Cells were incubated for 20 min at 37 °C with 1 μM FerroOrange working solution before fluorescent image acquisition by an EVOS M5000 microscope (Thermo Scientific, Rockford, IL, USA). Fluorescence intensity was recorded by a BD Accuri C6 cytometer (BD Biosciences, San José, CA, USA), and analysis was carried out by FlowJo V10 software (WilliamsonWay, Ashland, OR, USA).