Cited by 151Autophagy in Disease and TherapyCellular transport and secretionCalcium signaling and nucleotide metabolism

A novel quantitative flow cytometry-based assay for autophagy

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Eng, Kai Er, Panas, Marc D., Hedestam, Gunilla B. Karlsson, McInerney, Gerald M.·2010·Autophagy

Abstract

Autophagy is a cellular degradation process with an increasingly recognised importance in many biological pathways such as nutrient sensing, stress responses and development. We present a straightforward assay for autophagy which combines the sensitivity of the EGFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the non-autophagosome associated EGFP-LC3-I form of the protein, flow cytometry can be used to measure total fluorescence of saponin extracted HOS-EGFP-LC3 cells as a measure of the levels of autophagosome associated EGFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to differentiate between constitutive and induced autophagy and to quantify the changes in flux of the system. Moreover, using direct antibody staining for the endogenous LC3 protein, we have extended this assay to the detection of autophagosome formation in non-transfected cells.

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Authors
Eng, K. E., Panas, M. D., Hedestam, G. B. K., & McInerney, G. M.
Affiliations
Karolinska Institutet (SE)
Corresponding author
McInerney, Gerald M.
Venue
Autophagy
Published
2010-07
Volume
6
Issue
5
Pages
634-641
ISSN
  • 1554-8627 (ISSN-L)
  • 1554-8635
OpenAlex
W2026312959
Cited by
151
References
19
Citations by year
2026: 3 · 2025: 2 · 2024: 3 · 2023: 6 · 2022: 6 · 2021: 11 · 2020: 17 · 2019: 14 · 2018: 8 · 2017: 17 · 2016: 14 · 2015: 12 · 2014: 13 · 2013: 7 · 2012: 12
FWCI
5.83
Type
journal article
Language
en
Open access
Locations
  • Autophagy (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Karolinska Institutet
  • RIKEN
Topics
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
Keywords
autophagosome formation, saponin extraction, autophagic flux, autophagy inhibitors, flow cytometry quantification
MeSH terms
Animals, Autophagy, Biological Assay, Chloroquine, Flow Cytometry, Humans, Microtubule-Associated Proteins, Saponins, Intracellular Space, Green Fluorescent Proteins, Mice

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