<p class="first" id="d7717146e7782">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.
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.
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