Protocol for performing and analyzing a live-cell imaging-based microglia phagocytosis assay using AI on the Olympus ScanR system

Various neurological disorders show dysregulation of microglial function, including phagocytosis, necessitating accurate and efficient analysis. Here, we present a protocol using our custom-trained neural network (AIstain) in the Olympus ScanR environment for easy-to-use workflows of microglial phagocytosis assays. We describe steps for preparing microglia and setting up the microscope. We then explain the workflow of live-cell imaging and usage of AI in the data analysis. This protocol facilitates the preparation of phagocytosis assays and improves accuracy in data analysis. For complete details on the use and execution of this protocol, please refer to Zähringer et al.¹.