Multiplexed quantification of autophagic flux by imaging flow cytometry.

Abstract

Imaging flow cytometry allows for the quantitative assessment of fluorescent signals at the subcellular level. Here, we describe the use of a biosensor cell line, namely, U2OS osteosarcoma cells equipped with a fusion protein comprising monomeric red fluorescent protein (mRFP), green fluorescent protein (GFP) and microtubule-associated proteins 1A/1B light chain 3B (best known as LC3), for the assessment of autophagic flux by imaging flow cytometry. We detail all analysis tools required to distinguish autophagosomes (that emit both a red and a green fluorescence) and autolysosomes (that emit a red fluorescence, yet lose the green fluorescent signal) and to quantitate autophagic flux in a convenient fashion.

• Authors: Léa Montégut, Hui Chen, Gerasimos Anagnostopoulos, Sabrina Spaggiari, Oliver Kepp, Maria Chiara Maiuri, Guido Kroemer, Isabelle Martins
• Journal: Methods in cell biology (Methods Cell Biol) Vol. 165 Pg. 59-71 ( 2021) ISSN: 0091-679X [Print] United States
• PMID: 34311871 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2021 Elsevier Inc. All rights reserved.

Chemical References

• Microtubule-Associated Proteins
• Green Fluorescent Proteins

Topics

• Autophagosomes
• Autophagy
• Flow Cytometry
• Green Fluorescent Proteins (genetics)
• Humans
• Lysosomes
• Microtubule-Associated Proteins