A Ferric Ammonium Citrate-Based Model of Iron Overload With Ferroptosis-Associated Readouts In C2C12 Myoblasts

Ferroptosis is an iron-dependent form of regulated cell death implicated in aging and degenerative disease. We previously found that aged muscle stem cells accumulate intracellular iron and undergo ferroptotic death upon activation. Here, we describe a simple in vitro model that uses ferric ammonium citrate to generate iron overload in murine C2C12 myoblasts. Ferric ammonium citrate treatment increased intracellular iron burden, as shown by elevated ferritin heavy- and light-chain transcripts, increased ferritin protein, and enhanced signal from a live-cell labile iron dye. Ferric ammonium citrate (FAC) exposure also produced molecular changes associated with ferroptosis, including increased Slc40a1 and Hmox1 expression and reduced glutathione peroxidase 4 protein. The FAC-associated decrease in glutathione peroxidase 4 was partially reversed by deferoxamine. At 12 hours, cytotoxic ferric ammonium citrate reduced cell viability, and this effect was rescued by ferrostatin-1, consistent with a ferroptosis-sensitive component of cell death. At later time points, increased variability and reduced assay dynamic range limit the interpretability of viability measurements. Thus, this protocol provides a rapid and scalable model of ferric ammonium citrate-mediated iron loading that can be used to study ferroptosis-associated responses and test iron- or ferroptosis-modifying interventions in muscle cells.