Evolving methodologies for identification and differentiation of regulated cell death modalities

Cell death is a crucial evolutionary adaptation for multicellular organisms through which they can systematically eliminate cells that are no longer needed, potentially harmful, or are damaged beyond repair. Over the past few decades, our understanding of the cell death mechanisms has expanded significantly revealing a diverse, and interconnected array of regulated cell death (RCD) pathways that includes apoptosis, necroptosis, pyroptosis, cuproptosis etc. While the complexities of these pathways have incrementally increased with the evolution of multicellularity, many core components associated with cell death have remained conserved. This points towards the essential function of cell death in maintenance of homeostasis at the cellular, organismal and individual level. It is thus not a surprise that their dysregulation can manifest in the form of several pathologies. Therefore, the ability to accurately detect and distinguish different forms of cell death is essential not only for advancing our understanding of the fundamental cellular and molecular processes but also for elucidating their role in disease pathogenesis, where their dysregulation contributes to various pathological conditions. However, detecting and differentiating various forms of cell death is a challenging task. Since there are multiple cell death modalities, many of their characteristics overlap, such as a condensed nucleus being observed in both secondary necrosis and apoptosis. Further, a cell can undergo more than one kind of cell death simultaneously, a process known as "cell death continuum" further complicating detection and classification. This chapter provides an overview of the conventional methods used for detecting cell death, highlighting both probe-based and non-probe-based techniques. Recent advancements in high-throughput strategies, AI based predictive modelling and other such novel techniques that offer greater specificity in cell death characterization are particularly emphasized.