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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 thereisadifferencebetweenmeasurementsthatmonitorthenumbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes)atanystageoftheautophagicprocessversusthose 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 fromstimulithatincreaseautophagicactivity,definedasincreased autophagyinductioncoupledwithincreaseddeliveryto,anddegradationwithin,lysosomes(inmosthighereukaryotesandsomeprotists such as Dictyostelium) or the vacuole (in plants and fungi). In otherwords,itisespeciallyimportantthatinvestigatorsnewtothe field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases,autophagosomesaccumulatebecauseofablockintrafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reductionindegradativeactivity.Itisworthemphasizingherethat lysosomaldigestionisastageofautophagyandevaluatingitscompetence is a crucial part of the evaluation of autophagic flux, or completeautophagy. 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 autophagyrelated 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.

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