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

ABSTRACT: Summary 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). Graphical abstract Highlights • Detailed protocol to monitor autophagy in live cells based on turboGFP-LC3B dynamics• Stable expression of turboGFP-LC3B as a reporter for automated autophagy measurement• Rapid quantification of effects of starvation in live cells over time• Use of high-throughput microscopy to monitor metabolic response of cells to stress 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.

SUBMITTER: Nowosad A

PROVIDER: S-EPMC8605097 | biostudies-literature |

REPOSITORIES: biostudies-literature

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