Multiplexed quantification of autophagic flux by imaging flow cytometry

Léa Montégut; Hui Chen; Gerasimos Anagnostopoulos; Sabrina Spaggiari; Oliver Kepp; Maria Chiara Maiuri; Guido Kroemer; Isabelle Martins

doi:10.1016/bs.mcb.2021.02.005

Multiplexed quantification of autophagic flux by imaging flow cytometry

Methods Cell Biol. 2021:165:59-71. doi: 10.1016/bs.mcb.2021.02.005. Epub 2021 Mar 27.

Authors

Léa Montégut¹, Hui Chen¹, Gerasimos Anagnostopoulos¹, Sabrina Spaggiari², Oliver Kepp², Maria Chiara Maiuri², Guido Kroemer³, Isabelle Martins⁴

Affiliations

¹ Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Université Paris Sud/Paris XI, Faculté de médecine, Kremlin-Bicêtre, France.
² Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
³ Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Pôle de Biologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France; Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China; Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Physiology, University Complutense of Madrid, Madrid, Spain. Electronic address: [email protected].
⁴ Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France. Electronic address: [email protected].

PMID: 34311871
DOI: 10.1016/bs.mcb.2021.02.005

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.

Keywords: Autophagic flux; Imaging flow cytometry; Multiplexed quantification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Autophagosomes*
Autophagy*
Flow Cytometry
Green Fluorescent Proteins / genetics
Humans
Lysosomes
Microtubule-Associated Proteins

Substances

Microtubule-Associated Proteins
Green Fluorescent Proteins