Research in autophagy continues to accelerate,1and 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.2,3There 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.
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an autophagy inhibitor or resulting from Atg gene knockdowns). oversight or any other reason, could not be included on this manu-Collectively, we strongly recommend the use of multiple assays script. This work was supported by National Institutes of Health whenever possible, rather than relying on the results from a single Public Health Service grant GM53396 to D.J.K. Due to space and method. other limitations, it is not possible to include all other sources of As a final reminder, we stated at the beginning of this review financial support. that this set of guidelines is not meant to be a formulaic set of References rules, because the appropriate assays depend in part on the question 1. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a being asked and the system being used. Rather, these guidelines are decade. Nat Rev Mol Cell Biol 2007; 8:931-7. presented primarily to emphasize key issues that need to be addressed human. Autophagy 2007; 3:181-206.2.Klionsky DJ, Cuervo AM, Seglen PO. Methods for monitoring autophagy from yeast to Bioscience. such as the difference between measuring autophagy components, 3. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004; 36:2491- and flux or substrate clearance; they are not meant to constrain imag-502. inative approaches to monitor autophagy. Hopefully, new methods tocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine. Exp Cell Kovács AL, Reith A, Seglen PO. Accumulation of autophagosomes after inhibition of hepa- for monitoring autophagy will continue to be developed, and new Res 1982; 137:191-201. findings may alter our view of the current assays. For example, one Seglen PO. Regulation of autophagic protein degradation in isolated liver cells. In: area that shows promise is the use of nanoparticles as tools for moni-Academic Press, 1987:369-414.Glaumann H, Ballard FJ, eds. Lysosomes: Their Role in Protein Breakdown. London: toring autophagy,208 as they could be used in EM (e.g., providing de Duve C, Wattiaux R. Functions of lysosomes. Annu Rev Physiol 1966; 28:435-92. contrast by using different sizes and shapes of nanoparticles) or tion. Trends Cell Biol 1994; 4:139-43.Dunn WA, Jr. Autophagy and related mechanisms of lysosome-mediated protein degrada- to follow autophagLic faluxn ind liveinsg c ells (e.g., relying on the stable Gordon PB, Seglen PO. Prelysosomal convergence of autophagic and endocytic pathways. fluorescence of quantum dots) allowing the tracking of autophago-Biochem Biophys Res Commun 1988; 151:40-7. somes and amphisomes. Similar to the process of autophagy, this Dunn WA, Jr. Studies on the mechanisms of autophagy: formation of the autophagic vacu- is a dynamic field, and we need to remain flexible in the standards Ishihara N, Hamasaki M, Yokota S, Suzuki K, Kamada Y, Kihara A, Yoshimori T, Noda ole. J Cell Biol 1990; 110:1923-33. we apply. T, Ohsumi Y. Autophagosome requires specific early Sec proteins for its formation and