Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1

Research output: Contribution to journalReview articleContributedpeer-review


  • University of Michigan, Ann Arbor
  • Ain Shams University
  • IRCCS Istituto Europeo di Oncologia - Milano
  • Johannes Gutenberg University Mainz
  • Medical University of Graz
  • Pasteur Institute of Iran
  • Leibniz Institute of Plant Biochemistry
  • Hebrew University of Jerusalem
  • Danish Cancer Society
  • University of Copenhagen
  • UiT The Arctic University of Norway
  • Hospital Universitario de Canarias
  • University of California at Davis
  • University of Toronto
  • Innsbruck Medical University
  • University of Calabria
  • National Institutes of Health
  • Ben-Gurion University of the Negev
  • University of Alabama at Birmingham
  • Inflammation Research Center San Diego
  • University of Palermo
  • KU Leuven
  • Indian Institute of Technology Ropar
  • University of Dundee
  • University of Texas Medical Branch at Galveston
  • Colby College
  • University of Maryland, Baltimore
  • Universidad Mayor
  • Lund University
  • Nihon University
  • Koc University
  • University of Crete
  • Institute of Cancer Research
  • Gulf Medical University
  • University of Sydney
  • Naval Res Lab, United States Department of Defense, United States Navy, Naval Research Laboratory
  • McGill University
  • Technische Universität Dresden
  • University of Seville
  • University of Rome La Sapienza
  • Johns Hopkins University
  • Rajshahi University
  • Islamic University of Gaza
  • University of Manitoba
  • University of Barcelona
  • The Francis Crick Institute
  • Cleveland Clinic Foundation
  • University of the Basque Country
  • Universidad de Buenos Aires
  • Wistar Institute
  • University of Porto


In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.


Original languageEnglish
Pages (from-to)1-382
Number of pages382
Issue number1
Publication statusPublished - 2021

External IDs

PubMed 33634751


Sustainable Development Goals

ASJC Scopus subject areas


  • Autophagosome, cancer, flux, LC3, lysosome, macroautophagy, neurodegeneration, phagophore, stress, vacuole