A high-throughput protocol for monitoring starvation-induced autophagy in real time in mouse embryonic fibroblasts

Ada Nowosad; Arnaud Besson

doi:10.1016/j.xpro.2021.100966

A high-throughput protocol for monitoring starvation-induced autophagy in real time in mouse embryonic fibroblasts

STAR Protoc. 2021 Nov 17;2(4):100966. doi: 10.1016/j.xpro.2021.100966. eCollection 2021 Dec 17.

Authors

Ada Nowosad^{1

2}, Arnaud Besson¹

Affiliations

¹ Molecular, Cellular and Developmental Biology Department (MCD), Centre de Biologie Intégrative (CBI), University of Toulouse, CNRS, UPS, 31062 Toulouse, France.
² Laboratory of Molecular Cancer Biology, VIB Center for Cancer Biology, KU Leuven, Leuven, Belgium.

Abstract

Autophagy measurement has been challenging due to the transient nature of autophagy vesicles, in which degradation of cargo occurs. Here, we present a protocol to monitor starvation-induced autophagy using a live high-throughput microscopy system in a fast and automated manner without the need for sample preparation. We provide a detailed protocol describing the generation of turboGFP-LC3B expressing mouse embryonic fibroblasts (MEFs), the measurement of autophagy over time and the analysis of data. For complete details on the use and execution of this protocol, please refer to Nowosad et al. (2020, 2021).

Keywords: Cell Biology; Cell-based Assays; High Throughput Screening; Microscopy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Autophagy / physiology*
Cell Culture Techniques
Cells, Cultured
Fibroblasts / cytology*
High-Throughput Screening Assays / methods*
Mice
Microscopy / methods*
Starvation