Characterization of a rat monoclonal antibody raised against ferroptotic cells

Ferroptosis is regulated, non-apoptotic cell death in which ferrous iron and lipid peroxidation products play essential roles. While the ferroptotic pathway is now becoming unveiled, it is difficult to determine its involvement in situ because no unique marker for ferroptotic cells is known. In this study, we report on raising a rat monoclonal antibody against mouse-derived Hepa 1-6 cells that had been cultivated in cystine-deprived media. Binding of the resulting antibody, designated as FerAb, increased during advancing ferroptosis which was caused, not only by cystine deprivation but also treatment with erastin or RSL3, while apoptotic cell death induced by a staurosporine treatment had no effect on the binding. The FerAb was found to bind to 4-hydroxy-2-nonenal (HNE)-modified bovine serum albumin, but no specific protein was detected in ferroptotic cells in an immunoblot analysis. These results indicate that non-proteinaceous, HNE-like structural moiety was part of the antigen for FerAb, although the binding profiles of FerAb to ferroptotic cells were different from those of the currently available anti-HNE antibody. Immunocytological detection revealed inhomogenous staining within cells and partial co-localization with peripheral mitochondria and other cellular components. FerAb was found to be applicable for ferroptotic cells in other mouse cells and cultured human cells that were examined. Thus, the properties of the rat monoclonal antibody FerAb established in this study promise to be useful for the characterization of ferroptotic cell death.