Lipid ROS measurement by spectral flow cytometry based on the ratiometric dye BODIPY™ C11 581/591

The detection and measurement of lipid reactive oxygen species (ROS) is a critical method widely applied in the study of ferroptosis, a recently defined form of iron-dependent programmed cell death characterized by excessive lipid peroxidation and subsequent membrane damage. As ferroptosis research expands into various pathophysiological processes and human diseases, including cancer, immunity and inflammation, and tissue injuries, there is an increasing need for robust methods to measure lipid ROS in heterogenous cell samples, such as in deep immune cell profiling. BODIPY™ C11 581/591 is a gold-standard ratiometric fluorescent dye that reports the levels of lipid ROS, and has been used in both mechanistic and applied studies demonstrating ferroptosis regulation across multiple cell types. However, its use has largely been confined to traditional flow cytometry due to its extensive spillover into multiple emission channels and its ratiometric properties, which complicate its application in spectral flow cytometry. Herein, we present solutions to these challenges and a comprehensive workflow for the optimization, unmixing, staining, and analysis required to multiplex lipid ROS detection using BODIPY™ C11 581/591 with high-parameter spectral flow cytometry panels. Our unmixing strategy represents a novel method for separating the fluorescent spectra of two forms of a ratiometric dye, which may also be applicable to other ratiometric dyes in spectral flow cytometry.