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

Daniel J. Klionsky, Life Sciences Institute and Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
Amal Kamal Abdel-Aziz, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.
Sara Abdelfatah, Johannes Gutenberg University, Institute of Pharmaceutical and Biomedical Sciences, Department of Pharmaceutical Biology, Mainz, Germany.
Mahmoud Abdellatif, Medical University of Graz, Division of Cardiology, Graz, Austria.
Asghar Abdoli, Pasteur institute of Iran, Department of Hepatitis and HIV, Tehran, Iran.
Steffen Abel, Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
Hagai Abeliovich, Hebrew University of Jerusalem, Department of Biochemistry and Food Science, Rehovot, Israel.
Marie H. Abildgaard, Danish Cancer Society Research Center, RNA and Autophagy Group, Copenhagen, Denmark.
Yakubu Princely Abudu, University of Tromsø - The Arctic University of Norway, Department of Medical Biology, Tromsø, Norway.
Abraham Acevedo-Arozena, Hospital Universitario de Canarias, Research Unit; CIBERNED and Universidad de La Laguna, ITB, Santa Cruz de Tenerife, Spain.
Iannis E. Adamopoulos, University of California, Davis, Division of Rheumatology, Allergy and Clinical Immunology, School of Medicine, Sacramento, CA, USA.
Khosrow Adeli, University of Toronto, Department of Biochemistry, Toronto, Ontario, Canada.
Timon E. Adolph, Medical University Innsbruck, Department of Medicine I, Innsbruck, Austria.
Annagrazia Adornetto, University of Calabria, Department of Pharmacy, Health and Nutritional Sciences, Arcavacata di Rende (CS), Italy.
Elma Aflaki, National Institutes of Health, National Eye institute, Protein Structure and Function Section, Bethesda, MD, USA.
Galila Agam, Ben-Gurion University of the Negev, Faculty of Health Sciences, Department of Clinical Biochemistry and Pharmacology, Beer-Sheva, Israel.
Anupam Agarwal, University of Alabama at Birmingham, Division of Nephrology and Birmingham VA Medical Center, Birmingham, AL, USA.
Bharat B. Aggarwal, Inflammation Research Center, San Diego, CA, USA.
Maria Agnello, University of Palermo, Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Palermo, Italy.
Patrizia Agostinis, Cell Death Research & Therapy (CDRT) Lab, Department of Cellular & Molecular Medicine, Center for Cancer Biology, VIB-KU Leuven, Leuven, Belgium.
Javed N. Agrewala, Indian Institute of Technology, Ropar, India.
Alexander Agrotis, University of Dundee, School of Life Sciences, MRC Protein Phosphorylation and Ubiquitylation Unit, Dundee, UK.
Patricia V. Aguilar, University of Texas Medical Branch, Department of Pathology, Galveston, TX, USA.
S Tariq Ahmad, Colby College, Department of Biology, Waterville, ME, USA.
Zubair M. Ahmed, University of Maryland School of Medicine, Departments of Otorhinolaryngology Head & Neck Surgery, Ophthalmology and Visual Science, Baltimore, MD, USA.
Ulises Ahumada-Castro, Universidad Mayor, Center for Integrative Biology, and Geroscience Center for Brain Health and Metabolism, Santiago, Chile.
Sonja Aits, Lund University, Department of Experimental Medical Science, Cell Death, Lysosomes and Artificial Intelligence Group, Lund, Sweden.
Shu Aizawa, Nihon University, College of Bioresource Sciences, Fujisawa, Kanagawa, Japan.
Yunus Akkoc, Koç University School of Medicine, Department of Molecular Biology and Genetics and KUTTAM Research Center for Translational Medicine, Istanbul, Turkey.
Tonia Akoumianaki, University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.
Hafize Aysin Akpinar, The Institute of Cancer Research, Cancer Therapeutics Unit, Sutton, UK.
Ahmed M. Al-Abd, Pharmaceutical Sciences Department, College of Pharmacy, Gulf Medical University, Ajman, United Arab Emirates.
Lina Al-Akra, University of Sydney, Department of Pathology and Bosch Institute, Sydney, NSW, Australia.


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 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.