Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of
assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in
yeast, but relatively few in other
model systems, and there is much
confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within,
lysosomes (in most higher eukaryotes) or the
vacuole (in plants and
fungi). Here, we present a set of guidelines for the
selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.
