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

Daniel J Klionsky, Amal Kamal Abdel-Aziz, Daniela Barilà, Matteo Bordi, Riccardo Calvani, Michelangelo Campanella, Silvia Campello, Silvia Carloni, Marco Castori, Francesco Cecconi, Simone Cenci, Susanna Chiocca, Valerio Chiurchiù, Emilio Clementi, Eliana M. Coccia, Valeria Crippa, Jessica Dal Col, Alessio Di Fonzo, Martina Di Rienzo, Mohamed ElgendyAntonio Facchiano, Francesco Facchiano, Gian Maria Fimia, Tullio Florio, Francesco Fornai, Paola Fortini, Carmela Fusco, Cecilia Gelfi, Valeria Gerbino, Giuseppe Ippolito, Ilaria Laface, Min Li, Min Li, Federica Limana, Walter Malorni, Angelo A. Manfredi, Fabrizio Marcucci, Sara Marinelli, Sara Martin, Emanuele Marzetti, Paola Matarrese, Domenico Mattoscio, Gianluigi Mazzoccoli, Giuseppe Merla, Lucia Micale, Enrico Milan, Vincenzo Mollace, Barbara Montico, Marina Mora, Sandra Moreno, Roberta Nardacci, Francesca Nazio, Annalisa Nobili, Elena Ortona, Michela Palmieri, Giuseppe Palmisano, Francesca Pentimalli, Anna Picca, Marina Pierdominici, Patrizia Rovere-Querini, Alessandra Ruggieri, Maria Russo, Marco Sandri, Stefano Santaguida, Francesca Scatozza, Maria Vittoria Schiaffino, Monica Schiappacassi, Sebastiano Sciarretta, Venturina Stagni, Flavie Strappazzon, Marco Tafani, Chiara Vantaggiato, Sonia Viganó, Maria Teresa Viscomi, Ilio Vitale, Cinzia Volonté, Yu Wang, Elena Ziviani

Research output: Contribution to journalArticlepeer-review


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
Publication statusPublished - 2021


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


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