Daniel J. KlionskyFabio C. AbdallaHagai AbeliovichRobert T. AbrahamAbraham Acevedo-ArozenaKhosrow AdeliLotta AgholmeMaria AgnelloPatrizia AgostinisJulio A. Aguirre-GhisoHyung Jun AhnOuardia Ait-MohamedSlimane Ait-Si-AliTakahiko AkematsuShizuo AkiraHesham M. Al-YounesMunir A. Al-ZeerMatthew L. AlbertRoger L. AlbinJavier Alegre-AbarrateguiMaria Francesca AleoMehrdad AlirezaeiAlexandru AlmasanMaylin Almonte-BecerrilAtsuo AmanoRavi AmaravadiShoba AmarnathAmal O. AmerNathalie Andrieu-AbadieVellareddy AnantharamDavid K. AnnShailendra Anoopkumar-DukieHiroshi AokiNadezda ApostolovaGiuseppe AranciaJohn P. ArisKatsuhiko AsanumaNana Y.O. AsareHisashi AshidaValerie AskanasDavid S. AskewPatrick AubergerMisuzu BabaSteven K. BackuesEric H. BaehreckeBen A. BahrXue Yuan BaiYannick BaillyRobert BaiocchiGiulia BaldiniWalter BalduiniAndrea BallabioBruce A. BamberEdward T.W. BamptonGábor BánhegyiClinton R. BartholomewDiane C. BasshamRobert C. BastHenri BatokoBoon Huat BayIsabelle BeauDaniel M. 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UniversityOIST Graduate UniversityVanderbilt Ingram Cancer CenterWeis Center for ResearchUniversity of ShizuokaINRA Centre de Recherche de Versailles-GrignonChosun UniversityIcahn School of Medicine at Mount SinaiKeio University School of MedicineToronto General Research Institute University of TorontoFondazione IRCCS Istituto Nazionale dei Tumori, MilanUniversity of VirginiaNational Institute of Biological Sciences, BeijingLeiden University Medical Center - LUMCUniversity of Newcastle Faculty of Medicine and Health SciencesPeking University Health Science CenterUniversity of California, BerkeleyGoteborgs UniversitetMax Planck Institut fur PsychiatrieCentre Hospitalier Universitaire de NiceIstituto Neurologico Mediterraneo Neuromed, PozzilliThe Cancer Institute of New JerseyUniversite de LyonPanepistimio KritisSanto Tomas UniversityJohannes Gutenberg Universitat MainzUniversity of Rzeszow2018-06-112018-06-112012-04-01Autophagy. Vol.8, No.4 (2012), 445-54415548635155486272-s2.0-84862295360https://repository.li.mahidol.ac.th/handle/20.500.14594/13771In 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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field. © 2012 Landes Bioscience.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyReviewSCOPUS10.4161/auto.19496