Understanding radiation-induced tissue modifications and cell death within the complex cellular architecture of the brain requires experimental systems that preserve tissue integrity and intercellular crosstalk. At the same time, microglial cells as primary regulators of primary and secondary inflammation in the brain are difficult to study in single cell cultures and it is crucial to investigate their behaviour in their complex tissue microenvironment. Rodent organotypic hippocampal slice cultures (OHSCs) and human patient-derived brain tumor cultures (PDTCs) offer physiologically relevant platforms for investigating the differential effects of X-ray and heavy-ion irradiation on glial as well as immune cell populations. Both models retain native cytoarchitecture, extracellular matrix composition, and functional cell-cell interactions, making them uniquely suited to study the spatial and temporal dynamics of inflammation or cell death in a multicellular context. Here, we present a standardized methodology for preparing, irradiating, and assessing PDTCs, employing multimodal readouts as cytokine measurement of the supernatant, cell death, microglia morphology and function. Novel molecular analyses are possible upon certain considerations and may alter our understanding of species differences and help to distinguish cell death pathways to attribute damage to specific cell types. Standardization of these protocols across model systems is essential for generating reproducible, comparative data while preserving the inherent complexity that underlies intercellular communication and coordinated responses to injury. This approach enables translationally relevant insight into the mechanisms of radiation-induced brain injury to develop strategies for neuroprotection and treatment in clinical settings.
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.
Macrophages are key innate immune effector cells capable of responding to microbial factors such as lipopolysaccharide (LPS) through activation of inflammatory pathways. However, repeated LPS exposure can lead to endotoxin tolerance, a reprogrammed state where macrophages suppress pro-inflammatory cytokine production while preserving antimicrobial functions. The reduction in inflammatory responses during endotoxin tolerance serves to preserve tissue integrity and macrophage viability, although it can also result in immunosuppression, as observed in sepsis. An enhanced understanding of the molecular mechanisms which govern endotoxin tolerance in macrophages could unlock new ways to modulate innate immunity. Here we provide detailed protocols for inducing and measuring tolerance in murine bone marrow-derived macrophages (BMDMs) and human PMA-differentiated THP-1 macrophages, using ELISA-based quantification of IL-1β and IL-6 as functional readouts. These methods provide robust in vitro systems to study endotoxin tolerance.
