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Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

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Autophagy, 4 (2): 151-75 (February 2008)Klionsky, Daniel J Abeliovich, Hagai Agostinis, Patrizia Agrawal, Devendra K Aliev, Gjumrakch Askew, David S Baba, Misuzu Baehrecke, Eric H Bahr, Ben A Ballabio, Andrea Bamber, Bruce A Bassham, Diane C Bergamini, Ettore Bi, Xiaoning Biard-Piechaczyk, Martine Blum, Janice S Bredesen, Dale E Brodsky, Jeffrey L Brumell, John H Brunk, Ulf T Bursch, Wilfried Camougrand, Nadine Cebollero, Eduardo Cecconi, Francesco Chen, Yingyu Chin, Lih-Shen Choi, Augustine Chu, Charleen T Chung, Jongkyeong Clarke, Peter G H Clark, Robert S B Clarke, Steven G Clave, Corinne Cleveland, John L Codogno, Patrice Colombo, Maria I Coto-Montes, Ana Cregg, James M Cuervo, Ana Maria Debnath, Jayanta Demarchi, Francesca Dennis, Patrick B Dennis, Phillip A Deretic, Vojo Devenish, Rodney J Di Sano, Federica Dice, J Fred Difiglia, Marian Dinesh-Kumar, Savithramma Distelhorst, Clark W Djavaheri-Mergny, Mojgan Dorsey, Frank C Droge, Wulf Dron, Michel Dunn, William A Jr Duszenko, Michael Eissa, N Tony Elazar, Zvulun Esclatine, Audrey Eskelinen, Eeva-Liisa Fesus, Laszlo Finley, Kim D Fuentes, Jose M Fueyo, Juan Fujisaki, Kozo Galliot, Brigitte Gao, Fen-Biao Gewirtz, David A Gibson, Spencer B Gohla, Antje Goldberg, Alfred L Gonzalez, Ramon Gonzalez-Estevez, Cristina Gorski, Sharon Gottlieb, Roberta A Haussinger, Dieter He, You-Wen Heidenreich, Kim Hill, Joseph A Hoyer-Hansen, Maria Hu, Xun Huang, Wei-Pang Iwasaki, Akiko Jaattela, Marja Jackson, William T Jiang, Xuejun Jin, Shengkan Johansen, Terje Jung, Jae U Kadowaki, Motoni Kang, Chanhee Kelekar, Ameeta Kessel, David H Kiel, Jan A K W Kim, Hong Pyo Kimchi, Adi Kinsella, Timothy J Kiselyov, Kirill Kitamoto, Katsuhiko Knecht, Erwin Komatsu, Masaaki Kominami, Eiki Kondo, Seiji Kovacs, Attila L Kroemer, Guido Kuan, Chia-Yi Kumar, Rakesh Kundu, Mondira Landry, Jacques Laporte, Marianne Le, Weidong Lei, Huan-Yao Lenardo, Michael J Levine, Beth Lieberman, Andrew Lim, Kah-Leong Lin, Fu-Cheng Liou, Willisa Liu, Leroy F Lopez-Berestein, Gabriel Lopez-Otin, Carlos Lu, Bo Macleod, Kay F Malorni, Walter Martinet, Wim Matsuoka, Ken Mautner, Josef Meijer, Alfred J Melendez, Alicia Michels, Paul Miotto, Giovanni Mistiaen, Wilhelm P Mizushima, Noboru Mograbi, Baharia Monastyrska, Iryna Moore, Michael N Moreira, Paula I Moriyasu, Yuji Motyl, Tomasz Munz, Christian Murphy, Leon O Naqvi, Naweed I Neufeld, Thomas P Nishino, Ichizo Nixon, Ralph A Noda, Takeshi Nurnberg, Bernd Ogawa, Michinaga Oleinick, Nancy L Olsen, Laura J Ozpolat, Bulent Paglin, Shoshana Palmer, Glen E Papassideri, Issidora Parkes, Miles Perlmutter, David H Perry, George Piacentini, Mauro Pinkas-Kramarski, Ronit Prescott, Mark Proikas-Cezanne, Tassula Raben, Nina Rami, Abdelhaq Reggiori, Fulvio Rohrer, Barbel Rubinsztein, David C Ryan, Kevin M Sadoshima, Junichi Sakagami, Hiroshi Sakai, Yasuyoshi Sandri, Marco Sasakawa, Chihiro Sass, Miklos Schneider, Claudio Seglen, Per O Seleverstov, Oleksandr Settleman, Jeffrey Shacka, John J Shapiro, Irving M Sibirny, Andrei Silva-Zacarin, Elaine C M Simon, Hans-Uwe Simone, Cristiano Simonsen, Anne Smith, Mark A Spanel-Borowski, Katharina Srinivas, Vickram Steeves, Meredith Stenmark, Harald Stromhaug, Per E Subauste, Carlos S Sugimoto, Seiichiro Sulzer, David Suzuki, Toshihiko Swanson, Michele S Tabas, Ira Takeshita, Fumihiko Talbot, Nicholas J Talloczy, Zsolt Tanaka, Keiji Tanaka, Kozo Tanida, Isei Taylor, Graham S Taylor, J Paul Terman, Alexei Tettamanti, Gianluca Thompson, Craig B Thumm, Michael Tolkovsky, Aviva M Tooze, Sharon A Truant, Ray Tumanovska, Lesya V Uchiyama, Yasuo Ueno, Takashi Uzcategui, Nestor L van der Klei, Ida Vaquero, Eva C Vellai, Tibor Vogel, Michael W Wang, Hong-Gang Webster, Paul Wiley, John W Xi, Zhijun Xiao, Gutian Yahalom, Joachim Yang, Jin-Ming Yap, George Yin, Xiao-Ming Yoshimori, Tamotsu Yu, Li Yue, Zhenyu Yuzaki, Michisuke Zabirnyk, Olga Zheng, Xiaoxiang Zhu, Xiongwei Deter, Russell L GM53396/GM/NIGMS NIH HHS/United States R01 AG026389-01A2/AG/NIA NIH HHS/United States R01 AR055255-02/AR/NIAMS NIH HHS/United States R01 GM075061-03/GM/NIGMS NIH HHS/United States Research Support, N.I.H., Extramural Review United States Autophagy Autophagy. 2008 Feb 16;4(2):151-75. Epub 2007 Nov 21..

Abstract

Research in autophagy continues to accelerate,(1) and 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.(2,3) There 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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