# Protocol for the quantification of digestive exophagy in cell culture

## TL;DR

This paper provides a detailed protocol for measuring how microglia partially degrade large Alzheimer’s amyloid-beta aggregates outside the cell in a lab setting.

## Contribution

A new, standardized method to quantify digestive exophagy by microglia in cell culture using fluorescence microscopy and image analysis.

## Key findings

- Steps are outlined for isolating microglial cells and preparing labeled amyloid-beta aggregates.
- Lysosomal exocytosis and extracellular degradation can be measured using quantitative fluorescence microscopy.
- Digital image analysis is recommended for accurate quantification of digestive exophagy.

## Abstract

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 Aβ1-42 aggregates•Steps to measure digestive exophagy of model Aβ1-42 aggregates by microglial cells•Steps to quantify lysosomal exocytosis and aggregate degradation by image analysis

Steps for extracting primary murine microglial cells from mouse neonates

Steps for preparing fluorescently labeled model Alzheimer’s fibrillar Aβ1-42 aggregates

Steps to measure digestive exophagy of model Aβ1-42 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.