An improved method for high-throughput quantification of autophagy in mammalian cells.

Scientific Reports
Lennart KoepkeKonstantin M J Sparrer

Abstract

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating ...Continue Reading

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Citations

Jun 4, 2021·Frontiers in Pharmacology·Bruna CaridiPatric Turowski
Jul 30, 2021·Nature Communications·Caterina Prelli BozzoFrank Kirchhoff
Aug 19, 2021·STAR Protocols·Maximilian HirschenbergerKonstantin Maria Johannes Sparrer
Nov 5, 2021·Frontiers in Pharmacology·Maximilian HirschenbergerPanagiotis Papatheodorou

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Datasets Mentioned

BETA
GSE147488

Methods Mentioned

BETA
flow-cytometry
transfections
PCR
transfection
flow cytometry
fluorescence-activated cell sorting
FACS
confocal microscopy
fluorescence microscopy
lipidation

Software Mentioned

Graph Pad Prism
FlowJo
ImageJ
MAGeCK
GeCKO
Trim Galore !

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