A novel quantitative flow cytometry-based assay for autophagy
Open accessAbstract
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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The membrane-associated LC3 analysis assay has been well-established and verified previously.
“This assay has been well-established and verified previously (Eng et al., 2010; He et al., 2009; Martinez et al., 2015).”
From LC3-Associated Endocytosis Facilitates β-Amyloid Clearance and Mitigates Neurodegeneration in Murine Alzheimer’s Disease (2019) · cites this paper as Eng et al., 2010
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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
- Publisher
- Informa UK Limited
- 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
- bronze
- doi.org
- 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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