@article { , title = {Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).}, abstract = {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 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 versus those 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 from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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.}, doi = {10.1080/15548627.2015.1100356}, eissn = {1554-8635}, issn = {1554-8627}, issue = {1}, journal = {Autophagy}, note = {School: sch\_lss\_2015}, pages = {1-222}, publicationstatus = {Published}, publisher = {Taylor \& Francis}, url = {http://researchrepository.napier.ac.uk/id/eprint/9656}, volume = {12}, keyword = {610 Medicine & health, 616 Diseases, R1 Medicine (General), LC3, autolysosome, autophagosome, chaperone-mediated autophagy, flux, lysosome, macroautophagy, phagophore, stress, vacuole}, year = {2016}, author = {Abdelmohsen, Kotb and Abe, Akihisa and Abedin, Md Joynal and Abeliovich, Hagai and Acevedo Arozena, Abraham and Adachi, Hiroaki and Adams, Christopher M and Adams, Peter D and Adeli, Khosrow and Adhihetty, Peter J and Adler, Sharon G and Agam, Galila and Agarwal, Rajesh and Aghi, Manish K and Agnello, Maria and Agostinis, Patrizia and Aguilar, Patricia V and Aguirre-Ghiso, Julio and Airoldi, Edoardo M and Ait-Si-Ali, Slimane and Akematsu, Takahiko and Akporiaye, Emmanuel T and Al-Rubeai, Mohamed and Albaiceta, Guillermo M and Albanese, Chris and Albani, Diego and Albert, Matthew L and Aldudo, Jesus and Algül, Hana and Alirezaei, Mehrdad and Alloza, Iraide and Almasan, Alexandru and Almonte-Beceril, Maylin and Alnemri, Emad S and Alonso, Covadonga and Altan-Bonnet, Nihal and Altieri, Dario C and Alvarez, Silvia and Alvarez-Erviti, Lydia and Alves, Sandro and Amadoro, Giuseppina and Amano, Atsuo and Amantini, Consuelo and Ambrosio, Santiago and Amelio, Ivano and Amer, Amal O and Amessou, Mohamed and Amon, Angelika and An, Zhenyi and Anania, Frank A and Andersen, Stig U and Andley, Usha P and Andreadi, Catherine K and Andrieu-Abadie, Nathalie and Anel, Alberto and Ann, David K and Anoopkumar-Dukie, Shailendra and Antonioli, Manuela and Aoki, Hiroshi and Apostolova, Nadezda and Aquila, Saveria and Aquilano, Katia and Araki, Koichi and Arama, Eli and Aranda, Agustin and Araya, Jun and Arcaro, Alexandre and Arias, Esperanza and Arimoto, Hirokazu and Ariosa, Aileen R and Armstrong, Jane L and Arnould, Thierry and Arsov, Ivica and Asanuma, Katsuhiko and Askanas, Valerie and Asselin, Eric and Atarashi, Ryuichiro and Atherton, Sally S and Atkin, Julie D and Attardi, Laura D and Auberger, Patrick and Auburger, Georg and Aurelian, Laure and Autelli, Riccardo and Avagliano, Laura and Avantaggiati, Maria Laura and Avrahami, Limor and Awale, Suresh and Azad, Neelam and Bachetti, Tiziana and Backer, Jonathan M and Bae, Dong-Hun and Bae, Jae-sung and Bae, Ok-Nam and Bae, Soo Han and Baehrecke, Eric H and Baek, Seung-Hoon and Baghdiguian, Stephen and Bagniewska-Zadworna, Agnieszka and Klionsky, Daniel J and Stevens, Craig} }