Tiny Organisms, Big Advantages: Exploring Membrane Damage Responses Using <i>Dictyostelium discoideum</i> in <scp>High‐Throughput Single‐Cell</scp> Analyses

The amoeba Dictyostelium discoideum (Dd) is a commonly used model for studying bacterial infection due to the high conservation of the cell-autonomous defense pathways with animal phagocytes. Using Dd as a single-cell model organism, a robust high-content (HC)-based assay is developed that allow as high throughput imaging and quantitative analysis of cellular responses to bacteria-induced and L-Leucyl-L-Leucine methyl ester (LLOMe)-induced membrane damage. This chapter focuses on optimized live imaging protocols to track LLOMe-mediated endolysosomal damage and repair. Achieving optimal spatial and temporal resolutions in HC is challenging, necessitating choices and optimizations. The chapter presents two examples that demonstrate the versatility/adaptability of the protocol from both experimental and analytical perspectives. This protocol can also be used to monitor cellular responses to other sterile damaging agents and pathogens in various cell types.