PROJECT SUMMARY Necroptosis is a genetically encoded necrotic cell death that is overactivated in many pro-inflammatory human diseases, including cancer, inflammatory bowel disease, liver injury, pancreatitis, neurodegenerative disorders, and a diverse range of viral, bacterial, and fungal infections. The necroptosis signaling cascade is mediated by the sequential activation of RIPK1 and RIPK3 kinases downstream of pro-inflammatory ligands such as TNF or microbe-associated molecules. MLKL (Mixed Lineage Kinase Like protein) is a pseudokinase that tetramerizes upon phosphorylation by RIPK3 to form water-permeable pores that drive cell membrane rupture. The parent award has been given for investigating the molecular mechanisms of programmed necrosis execution, focusing on the regulation of MLKL by upstream kinases and E3 ubiquitin ligases. Current methods for accurate real-time quantification of cell death execution kinetics are labor-intensive, expensive, and low-throughput. The differentiation between late apoptotic and necrotic cell death is challenging, complicating cell death data analysis and interpretation. To this end, within the scope of the R35, we request the Incucyte® S3 live-cell imaging system for real-time and reliable quantification of cell death kinetics. This system will enable us to perform label-free quantification of cell death, using machine-learning algorithms of the system, thereby bypassing any need for fluorescent probes, allowing us to perform cell death quantitation in a non-perturbing manner in experiments where only one subtype of cell death is observed. Incucyte® S3 will also allow us to perform cell death kinetics experiments over prolonged periods in real-time and differentiate between apoptosis and necroptosis. Moreover, this system will allow us to develop a method for monitoring the activation kinetics of kinases that mediate necroptosis using a fluorescent-protein-based reporter system to support our mechanistic studies within the scope of the R35 project. In summary, this sophisticated platform for continuous real-time monitoring of cell health and cell signaling will enable us with advanced, unique, and rigorous methodological capabilities for studying the molecular mechanisms of programmed necrosis execution, as it will allow us to perform highly efficient, quantitative, multiplexed, and cost-effective cell death quantification and kinase activity monitoring experiments. Thus, this equipment will significantly boost our efforts in deciphering the mechanisms that govern the execution of programmed necrosis and identifying novel druggable targets for therapeutic interventions aimed at necroptosis blockade in human inflammatory diseases where it is overactivated.