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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Bagniewska-Zadworna, Agnieszka; Aquilano, Katia; Arnould, Thierry; Armstrong, Jane L.; Ariosa, Aileen R.; Arimoto, Hirokazu; Arias, Esperanza; Arcaro, Alexandre; Araya, Jun; Aranda, Agustin; Arama, Eli; Araki, Koichi; Aquila, Saveria; Asanuma, Katsuhiko; Apostolova, Nadezda; Aoki, Hiroshi; Antonioli, Manuela; Anoopkumar-Dukie, Shailendra; Ann, David K.; Anel, Alberto; Andrieu-Abadie, Nathalie; Andreadi, Catherine K.; Andley, Usha P.; Andersen, Stig U.; Arsov, Ivica; Askanas, Valerie; An, Zhenyi; Azad, Neelam; Baghdiguian, Stephen; Baek, Seung Hoon; Baehrecke, Eric H.; Bae, Soo Han; Bae, Ok Nam; Bae, Jae Sung; Bae, Dong Hun; Backer, Jonathan M.; Bachetti, Tiziana; Awale, Suresh; Avrahami, Limor; Asselin, Eric; Avantaggiati, Maria Laura; Avagliano, Laura; Autelli, Riccardo; Aurelian, Laure; Auburger, Georg; Auberger, Patrick; Attardi, Laura D.; Atkin, Julie D.; Atherton, Sally S.; Atarashi, Ryuichiro; Anania, Frank A.; Amon, Angelika; Al-Rubeai, Mohamed; Agarwal, Rajesh; Akporiaye, Emman...


Agnieszka Bagniewska-Zadworna

Katia Aquilano

Thierry Arnould

Jane L. Armstrong

Aileen R. Ariosa

Hirokazu Arimoto

Esperanza Arias

Alexandre Arcaro

Jun Araya

Agustin Aranda

Eli Arama

Koichi Araki

Saveria Aquila

Katsuhiko Asanuma

Nadezda Apostolova

Hiroshi Aoki

Manuela Antonioli

Shailendra Anoopkumar-Dukie

David K. Ann

Alberto Anel

Nathalie Andrieu-Abadie

Catherine K. Andreadi

Usha P. Andley

Stig U. Andersen

Ivica Arsov

Valerie Askanas

Zhenyi An

Neelam Azad

Stephen Baghdiguian

Seung Hoon Baek

Eric H. Baehrecke

Soo Han Bae

Ok Nam Bae

Jae Sung Bae

Dong Hun Bae

Jonathan M. Backer

Tiziana Bachetti

Suresh Awale

Limor Avrahami

Eric Asselin

Maria Laura Avantaggiati

Laura Avagliano

Riccardo Autelli

Laure Aurelian

Georg Auburger

Patrick Auberger

Laura D. Attardi

Julie D. Atkin

Sally S. Atherton

Ryuichiro Atarashi

Frank A. Anania

Angelika Amon

Mohamed Al-Rubeai

Rajesh Agarwal

Emmanuel T. Akporiaye

Takahiko Akematsu

Slimane Ait-Si-Ali

Edoardo M. Airoldi

Julio Aguirre-Ghiso

Patricia V. Aguilar

Patrizia Agostinis

Maria Agnello

Manish K. Aghi

Galila Agam

Guillermo M. Albaiceta

Sharon G. Adler

Peter J. Adhihetty

Khosrow Adeli

Peter D. Adams

Christopher M. Adams

Hiroaki Adachi

Abraham Acevedo Arozena

Hagai Abeliovich

Md Joynal Abedin

Mohamed Amessou

Chris Albanese

Kotb Abdelmohsen

Dario C. Altieri

Amal O. Amer

Ivano Amelio

Santiago Ambrosio

Consuelo Amantini

Atsuo Amano

Giuseppina Amadoro

Sandro Alves

Lydia Alvarez-Erviti

Silvia Alvarez

Nihal Altan-Bonnet

Diego Albani

Covadonga Alonso

Emad S. Alnemri

Maylin Almonte-Beceril

Alexandru Almasan

Iraide Alloza

Mehrdad Alirezaei


Jesus Aldudo

Matthew L. Albert

Akihisa Abe

... et al.

... >100 authors

Daniel J. Klionsky


In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.


Bagniewska-Zadworna, A., Baghdiguian, S., Baek, S. H., Baehrecke, E. H., Bae, S. H., Bae, O. N., …>100 authors, .. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy, 12(1), 1-222.

Journal Article Type Review
Acceptance Date Sep 22, 2015
Online Publication Date Jan 21, 2016
Publication Date Jan 21, 2016
Journal Autophagy
Print ISSN 1554-8627
Electronic ISSN 1554-8635
Publisher Taylor & Francis
Peer Reviewed Peer Reviewed
Volume 12
Issue 1
Pages 1-222
Public URL
Publisher URL