Protocol for the quantification of digestive exophagy in cell culture

Microglia use digestive exophagy to partially degrade, extracellularly, Alzheimer’s amyloid-beta aggregates that are too large to be phagocytosed. Here, we present a protocol to quantify this mechanism in cell culture. We describe steps for extracting primary microglial cells and preparing amyloid-beta model aggregates. We then detail procedures for measuring lysosomal exocytosis toward, and extracellular degradation of, these deposits using quantitative fluorescence microscopy. We also provide guidance on quantifying the data using digital image analysis. For complete details on the use and execution of this protocol, please refer to Jacquet et al. 1 • Steps for extracting primary murine microglial cells from mouse neonates • Steps for preparing fluorescently labeled model Alzheimer’s fibrillar Aβ 1-42 aggregates • Steps to measure digestive exophagy of model Aβ 1-42 aggregates by microglial cells • Steps to quantify lysosomal exocytosis and aggregate degradation by image analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Microglia use digestive exophagy to partially degrade, extracellularly, Alzheimer’s amyloid-beta aggregates that are too large to be phagocytosed. Here, we present a protocol to quantify this mechanism in cell culture. We describe steps for extracting primary microglial cells and preparing amyloid-beta model aggregates. We then detail procedures for measuring lysosomal exocytosis toward, and extracellular degradation of, these deposits using quantitative fluorescence microscopy. We also provide guidance on quantifying the data using digital image analysis.