Guidelines for the use and interpretation of assays for monitoring autophagy

1. Autophagy. 2012 Apr;8(4):445-544.

Guidelines for the use and interpretation of assays for monitoring autophagy.

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Comment in
Cardiovasc Pathol. 2014 Jul-Aug;23(4):251.

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.

DOI: 10.4161/auto.19496
PMCID: PMC3404883
PMID: 22966490 [Indexed for MEDLINE]

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