Guidelines for the use and interpretation of assays for monitoring autophagy

Guidelines for the use and interpretation of assays for monitoring autophagy
Daniel J. KlionskyHagai AbeliovichRobert T. AbrahamAbraham Acevedo-ArozenaKhosrow AdeliLotta AgholmeMaria AgnelloPatrizia AgostinisJulio A. Aguirre-Ghiso안형준Ouardia Ait-MohamedSlimane Ait-Si-AliTakahiko AkematsuShizuo AkiraHesham M. Al-YounesMunir A. Al-ZeerMatthew L. AlbertRoger L. AlbinJavier Alegre-AbarrateguiMaria Francesca AleoMehrdad AlirezaeiAlexandru AlmasanMaylin Almonte-BecerrilAtsuo AmanoRavi AmaravadiShoba AmarnathAmal O. AmerNathalie Andrieu-AbadieVellareddy AnantharamDavid K. AnnShailendra Anoopkumar-DukieHiroshi AokiNadezda ApostolovaGiuseppe AranciaJohn P. ArisKatsuhiko AsanumaNana Y.O. AsareHisashi AshidaValerie AskanasDavid S. AskewPatrick AubergerMisuzu BabaSteven K. BackuesEric H. BaehreckeBen A. BahrXue-Yuan BaiYannick BaillyRobert BaiocchiGiulia BaldiniWalter BalduiniAndrea BallabioBruce A. BamberEdward T.W. BamptonGabor BanhegyiClinton R. BartholomewDiane C. BasshamRobert C. Bast, Jr.Henri BatokoBoon-Huat BayIsabelle BeauDaniel M. BechetThomas J. BegleyChristian BehlChristian BehrendsSoumeya BekriBryan BellaireLinda J. BendallLuca BenettiLaura BerliocchiHenri BernardiFrancesca BernassolaSebastien BesteiroIngrid Bhatia-KissovaXiaoning BiMartine Biard-PiechaczykJanice S. BlumLawrence H. BoisePaolo BonaldoDavid L. BooneBeat C. BornhauserKarina R. BortoluciIoannis BossisFrederic BostJean-Pierre BourquinPatricia BoyaMichael Boyer-GuittautPeter V. BozhkovNathan R. BradyClaudio BrancoliniAndreas BrechJay E. BrenmanAna BrennandEmery H. BresnickPatrick BrestDave BridgesMolly L. BristolPaul S. BrookesEric J. BrownJohn H. BrumellNicola Brunetti-Pierri
autophagy; assay; atg4; polymer; autolysosome; autophagosome; flux; LC3; lysosome; phagophore
Issue Date
VOL 8, NO 4, 445-544
There is no question that research on the topic of autophagy has expanded dramatically since the publication of the first set of guidelines.1 To help keep track of the field we have published a glossary of autophagy-related molecules and processes,883,884 and there are now databases that are specifically dedicated to autophagy including the Human Autophagy-dedicated Database (HADb; and the Autophagy Database ( With this continued influx of new researchers we think it is critical to try to define standards for the field. Accordingly, we have highlighted the uses and caveats of an expanding set of recommended methods for monitoring macroautophagy in a wide range of systems (Table 2). Importantly, investigators need to determine whether they are evaluating levels of early or late autophagic compartments, or autophagic flux. If the question being asked is whether a particular condition changes autophagic flux (i.e., the rate of delivery of autophagy substrates to lysosomes or the vacuole, followed by degradation), then assessment of steady-state levels of autophagosomes (e.g., by counting GFP-LC3 puncta, monitoring the amount of LC3-II without examining turnover, or by single time point electron micrographs) is not sufficient as an isolated approach. In this case it is also necessary to directly measure the flux of autophagosomes and/or autophagy cargo (e.g., in wild-type cells compared with autophagydeficient cells, the latter generated by treatment with an autophagy inhibitor or resulting from ATG gene knockdowns). Collectively, we strongly recommend the use of multiple assays whenever possible, rather than relying on the results from a single method.
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