Guidelines for the use and interpretation of assays for monitoring autophagy

  title={Guidelines for the use and interpretation of assays for monitoring autophagy},
  author={Daniel J. Klionsky and F{\'a}bio Camargo Abdalla and Hagai Abeliovich and Robert T. Abraham and Abraham Acevedo-Arozena and Khosrow Adeli and Lotta Agholme and Maria Carmen Agnello and Patrizia Agostinis and Julio A. Aguirre-Ghiso and Hyung Jun Ahn and Ouardia Ait-Mohamed and Slimane Ait-Si-Ali and Takahiko Akematsu and Shizuo Akira and Hesham Mahmoud Al-Younes and Munir A Al-Zeer and Matthew L. Albert and Roger L. Albin and Javier Alegre-Abarrategui and Maria Francesca Aleo and M. Alirezaei and Alexandru Almasan and Maylin Almonte-Becerril and Atsuo Amano and Ravi K. Amaravadi and Shoba Amarnath and Amal O. Amer and Nathalie Andrieu-Abadie and Vellareddy Anantharam and David Kong Ann and Shailendra Anoopkumar‐Dukie and Hiroshi Aoki and Nadezda Apostolova and Giuseppe Arancia and John P. Aris and Katsuhiko Asanuma and Nana Yaa Odamea Asare and Hisashi Ashida and Valerie Askanas and David S. Askew and Patrick Auberger and Misuzu Baba and Steven K. Backues and Eric H. Baehrecke and Ben A Bahr and Xue Bai and Yannick Bailly and Robert A Baiocchi and Giulia Baldini and Walter Balduini and Andrea Ballabio and Bruce A Bamber and Edward T. W. Bampton and G{\'a}bor B{\'a}nhegyi and Clinton R. Bartholomew and Diane C. Bassham and Robert C. Bast and Henri Batoko and Boon Huat Bay and Isabelle Beau and Daniel B{\'e}chet and Thomas J. Begley and Christian Behl and Christian Behrends and Soumeya Bekri and Bryan H Bellaire and Linda J Bendall and Luca Benetti and Laura Berliocchi and Henri Bernardi and Francesca Bernassola and S{\'e}bastien Besteiro and Ingrid Bhatia-Kiššová and Xiaoning Bi and Martine Biard-Piechaczyk and Janice S Blum and Lawrence H. Boise and Paolo Bonaldo and David L. Boone and Beat C. Bornhauser and Karina Ramalho Bortoluci and Ioannis Bossis and Fr{\'e}d{\'e}ric Bost and Jean-Pierre Bourquin and Patricia Boya and Michaël Boyer-Guittaut and Peter V. Bozhkov and Nathan Ryan Brady and Claudio Brancolini and Andreas Brech and Jay E. Brenman and Ana Brennand and Emery H. Bresnick and Patrick Brest and Dave Bridges and Molly L Bristol and Paul S. Brookes and Eric J. Brown and John H. Brumell and Nicola Brunetti‐Pierri and Ulf T. Brunk and Dennis E. Bulman and Scott J. Bultman and Geert Bultynck and Lena F. Burbulla and Wilfried Bursch and Jonathan P Butchar and Wanda Buzgariu and S{\'e}rgio Paulo Bydlowski and Ken Cadwell and Monika Cahova and Dongsheng Cai and Jiyang Cai and Qian Cai and Bruno Calabretta and Javier Calvo-Garrido and Nadine Camougrand and Michelangelo Campanella and Jenny Campos-Salinas and Eleonora Candi and Lizhi Cao and Allan B Caplan and Simon R. Carding and Sandra Morais Cardoso and Jennifer S Carew and Cathleen R Carlin and Virginie Carmignac and Leticia A M Carneiro and Serena Carra and Rosario Caruso and Giorgio Casari and Caty Casas and Roberta Castino and Eduardo Cebollero and Francesco Cecconi and Jean Celli and Hassan Chaachouay and Han-Jung Chae and Chee-Yin Chai and David C. Chan and Edmond Y. W. Chan and Raymond Chuen-Chung Chang and Chi‐Ming Che and Ching‐Chow Chen and Guang-Chao Chen and Guoqiang Chen and Min Chen and Quan Chen and Steve S.-L. Chen and Wenli Chen and Xi Chen and Xiangmei Chen and Xiequn Chen and Ye Guang Chen and Yingyu Chen and Yongqiang Chen and Yu-Jen Chen and Zhixiang Chen and Alan Cheng and Christopher H. K. Cheng and Yan Cheng and Heesun Cheong and Jae Ho Cheong and Sara Cherry and Russ Chess-Williams and Zelda H.Y. Cheung and Eric Chevet and Hui-Ling Chiang and Roberto Chiarelli and Tomoki Chiba and Lih-Shen Chin and Shih-Hwa Chiou and Francis V. Chisari and Chi Hin Cho and Dong-Hyung Cho and Augustine M K Choi and DooSeok Choi and Kyeong Sook Choi and Mary E. Choi and Salem Chouaib and Divaker Choubey and Vinay Choubey and Charleen T. Chu and Tsung-Hsien Chuang and Sheau-Huei Chueh and Taehoon Chun and Yong-Joon Chwae and Mee‐Len Chye and Roberto Ciarcia and Maria Rosa Ciriolo and M. J. Clague and Robert S. B. Clark and Peter G. H. Clarke and Robert Clarke and Patrice Codogno and Hilary A. Coller and Mar{\'i}a Isabel Colombo and Sergio Comincini and Maria Condello and Fabrizio Condorelli and Mark R. Cookson and Graham H. Coombs and Isabelle Coppens and Ram{\'o}n Corbal{\'a}n and Pascale Cossart and Paola Costelli and Safia Costes and Ana Coto-Montes and Eduardo Couve and Fraser P. Coxon and James Michael Cregg and Jose Luis Crespo and Marianne J Cronj{\'e} and Ana Mar{\'i}a Cuervo and Joseph J Cullen and Mark J Czaja and Marcello D’Amelio and Arlette Darfeuille‐Michaud and Lester M. Davids and Faith E. Davies and Massimo De Felici and John F. de Groot and Cornelis A. M. de Haan and Luisa De Martino and Angelo De Milito and Vincenzo De Tata and Jayanta Debnath and Alexei Degterev and Benjamin Dehay and Lea M. D. Delbridge and Francesca Demarchi and Yi Zhen Deng and J{\"o}rn Dengjel and Paul Dent and Donna Denton and Vojo Deretic and Shyamal Desai and Rodney J Devenish and Mario Di Gioacchino and Gilbert Di Paolo and Chiara Di Pietro and Guillermo D{\'i}az-Araya and In{\'e}s D{\'i}az-Laviada and Maria T Diaz-Meco and Javier D{\'i}az-Nido and Ivan Dikic and Savithramma P. Dinesh-Kumar and Wen-Xing Ding and Clark W. Distelhorst and Abhinav Diwan and Mojgan Djavaheri-Mergny and Svetlana Dokudovskaya and Zheng Dong and Frank C. Dorsey and Victor E Dosenko and James J. Dowling and Stephen J. Doxsey and Marl{\`e}ne Dreux and Mark E. Drew and Qiuhong Duan and Michel A Duchosal and Karen E. Duff and Isabelle Dugail and Madeleine Durbeej and Michael Duszenko and Charles L. Edelstein and Aimee L Edinger and Gustavo Egea and Ludwig Eichinger and N Tony Eissa and Suhendan Ekmekcioglu and Wafik S. El-Deiry and Zvulun Elazar and Mohamed Elgendy and Lisa M. Ellerby and Kai Er Eng and Anna-Mart Engelbrecht and Simone Engelender and Jekaterina Erenpreisa and Ricardo Escalante and Audrey Esclatine and Eeva-Liisa Eskelinen and Lucile Espert and Virginia Espina and Huizhou Fan and Jia Fan and Qiuyue Fan and Zhengri Fan and Shengyun Fang and Yongqi Fang and Manolis Fanto and Alessandro Fanzani and Thomas Farkas and Jean-Claude Farr{\'e} and Mathias Faure and Marcus Fechheimer and Carl G. Feng and Jian Feng and Qili Feng and You-ji Feng and L{\'a}szl{\'o} F{\'e}s{\"u}s and Ralph Feuer and Maria E Figueiredo-Pereira and Gian Maria Fimia and Diane C Fingar and Steven Finkbeiner and Toren Finkel and Kim D. Finley and Filomena Fiorito and Edward A. Fisher and Paul B Fisher and Marc Flajolet and Maria L. Florez-McClure and Salvatore Florio and Edward A. Fon and Francesco Fornai and F Fortunato and Rati Fotedar and Daniel H. Fowler and Howard S. Fox and Rodrigo Franco and Lisa B. Frankel and Marc Fransen and Jos{\'e} M. Fuentes and Juan Fueyo and Junko Fujii and Kozo Fujisaki and Eriko Fujita and Mitsunori Fukuda and Ruth Furukawa and Matthias Gaestel and Philippe Gailly and Małgorzata Gajewska and Brigitte Galliot and Vincent Galy and Subramaniam Ganesh and Barry Ganetzky and Ian G. Ganley and Fen-Biao Gao and George Fu Gao and Jinming Gao and Lorena Garc{\'i}a and Guillermo Garcia-Manero and Mikel Garcia-Marcos and Marjan Garmyn and Andrei L Gartel and Evelina Gatti and Mathias Gautel and Thomas R Gawriluk and Matthew Gegg and Jiefei Geng and Marc Germain and Jason E. Gestwicki and David A. Gewirtz and Saeid Ghavami and Pradipta Ghosh and Anna Maria Giammarioli and Alexandra Giatromanolaki and Spencer B. Gibson and Robert Gilkerson and Michael L. Ginger and Henry N. Ginsberg and Jakub Gołąb and Michael S. Goligorsky and Pierre Golstein and Candelaria Gomez-Manzano and Ebru Goncu and C{\'e}line Gongora and Claudio Daniel Gonzalez and Ram{\'o}n Gonzalez and Cristina Gonz{\'a}lez-Est{\'e}vez and Rosa A. Gonz{\'a}lez-Polo and Elena Gonz{\'a}lez-Rey and Nikolai V Gorbunov and Sharon M. Gorski and Sandro Goruppi and Roberta A. Gottlieb and Devrim Gozuacik and Giovanna Elvira Granato and Gary Dean Grant and Kim N. Green and Ale{\vs} Gregorc and Fr{\'e}d{\'e}ric Gros and Charles Grose and Thomas W. Grunt and Philippe Gual and Jun-Lin Guan and Kun Liang Guan and Sylvie Guichard and Anna S. Gukovskaya and I. Ya. Gukovsky and Jan Gunst and {\AA}sa B Gustafsson and Andrew J. Halayko and Amber Hale and Sandra K. Halonen and Maho Hamasaki and Feng Han and Ting Han and Michael K. Hancock and Malene Hansen and Hisashi Harada and Masaru Harada and Stefan E Hardt and J. Wade Harper and Adrian L. Harris and James Harris and Steven D. Harris and Makoto Hashimoto and Jeffrey A Haspel and Shin-ichiro Hayashi and Lori A Hazelhurst and Congcong He and You-Wen He and M. J H{\'e}bert and Kim A. Heidenreich and Miep H Helfrich and G. Vignir Helgason and Elizabeth P. Henske and Brian Herman and Paul K. Herman and Claudio Hetz and Sabine Hilfiker and Joseph A. Hill and Lynne J. Hocking and Paul Hofman and Thomas G. Hofmann and J{\"o}rg H{\"o}hfeld and Tessa L Holyoake and Ming-huang Hong and David A. Hood and G{\"o}khan S. Hotamisligil and Ewout J. Houwerzijl and Maria H{\o}yer‐Hansen and Bingren Hu and Chien-An A. Hu and Hong Ming Hu and Ya-wei Hua and Canhua Huang and Ju Huang and Shengbing Huang and Wei‐Pang Huang and Tobias B. Huber and Won-Ki Huh and Tai-Ho Hung and Ted R. Hupp and Gang Min Hur and James B. Hurley and Sabah N A Hussain and Patrick J. Hussey and Jung Jin Hwang and Seungmin Hwang and Atsuhiro Ichihara and Shirin Ilkhanizadeh and Ken Inoki and Takeshi Into and Valentina Iovane and Juan Lucio Iovanna and Nancy Y. Ip and Yoshitaka Isaka and Hiroyuki Ishida and C. Isidoro and Ken-ichi Isobe and Akiko Iwasaki and Marta Izquierdo and Yotaro Izumi and Panu M. Jaakkola and Marja J{\"a}{\"a}ttel{\"a} and George R. Jackson and William T. Jackson and Bassam Janji and Marina Jendrach and Ju-Hong Jeon and Eui-Bae Jeung and Hongchi Jiang and Hongchi Jiang and Jean X. Jiang and Mingxi Jiang and Qing Jiang and Xuejun Jiang and Alberto Jim{\'e}nez and Meiyan Jin and Shengkan Jin and Cheol O. Joe and Terje Johansen and Daniel E Johnson and Gail V. W. Johnson and Nicola L. Jones and Bertrand Joseph and Suresh K. Joseph and Annie Margaretha Joubert and G{\'a}bor Juh{\'a}sz and Lucienne Juillerat-Jeanneret and Chang Hwa Jung and Yong-Keun Jung and Kai Kaarniranta and Allen Kaasik and Tomohiro Kabuta and Motoni Kadowaki and Shengzhou or sheng-zhou wu},
  pages={445 - 544}
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… 

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

There continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes, so it is important to update guidelines for monitoring autophagic activity in different organisms.

Direct quantification of autophagic flux by a single molecule-based probe

A new fluorescence probe is reported to directly demonstrate autophagic flux without being combined with lysosomal inhibitors and can be used to measure the cumulative effect of basal autophagy, applicable to high-throughput drug discovery as well as in vivo analysis.

Progress and Challenges in the Use of MAP1LC3 as a Legitimate Marker for Measuring Dynamic Autophagy In Vivo

This review emphasizes the methods applied to measure (macro-)autophagy in tissue samples and in vivo via a protein, which centrally intervenes in the autophagy pathway, the microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3), which is the most widely used marker and the first identified to associate with autophagosomal structures.

Blue-Print Autophagy in 2020: A Critical Review

This critical review provides an overview of the marine natural compounds, which have been reported to affect autophagy, and raises an important problem of standardization in the experimental design and data interpretation.

Measuring Autophagy in the Context of Cancer.

Three assays that are commonly used to evaluate autophagic flux in cells are outlined, including the determination ofLC3II formation and LC3II and p62 turnover by use of Western Blotting, quantification of LC3 puncta, and the measurement of autophagy flux using tandem labeled mCherry-GFP-LC3.

On the relevance of precision autophagy flux control in vivo – Points of departure for clinical translation

Key aspects that appear to contribute to stumbling blocks on the road toward clinical translation are highlighted and points of departure for reaching some of the desired goals are discussed, including techniques that are well aligned with achieving desirable spatiotemporal resolution.

Assays to Monitor Autophagy Progression in Cell Cultures

A review summarizing the most established assays used to monitor autophagy induction and progression in cell cultures is presented, in order to guide researchers in the selection of the most optimal solution for their experimental setup and design.

Methods to Measure Autophagy in Cancer Metabolism.

Some of the best-known and most widely used methods to study autophagy in vitro in the context of cancer, including transmission electron microscopy (TEM), detection and quantification of the autophile protein LC3 by western blot, and the use of GFP-LC3 to quantify puncta by fluorescence microscopy and tandem labeled RFP/mCherry-GFP- LC3 fluorescence microscopeopy to measure autophagic flux.

Methods to Measure Autophagy in Cancer Metabolism.

Some of the best-known and most widely used methods to study autophagy in vitro in the context of cancer, including transmission electron microscopy (TEM), detection and quantification of the autophile protein LC3 by western blot, and the use of GFP-LC3 to quantify puncta by fluorescence microscopy and tandem labeled RFP/mCherry-GFP- LC3 fluorescence microscopeopy to measure autophagic flux.



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

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 are presented.

Methodological considerations for assessing autophagy modulators: A study with calcium phosphate precipitates

It is shown that CPP induces autophagosome synthesis at early time-points, such as 4 h and 6 h, but at 24 h after treatment, the complex consequences of CPP suggest that it is best avoided as a transfection reagent in studies aiming to analyse Autophagy itself, or processes that are modulated by autophagy, like apoptosis.

Methods for Monitoring Autophagy from Yeast to Human

Current approaches that can be used to follow the overall process of autophagy, as well as individual steps, from yeast to human are discussed.

Autophagy: assays and artifacts

A critical overview of current methodologies to measure autophagy in cells and in animals is presented and the realization that it is not sufficient to monitor static levels of Autophagy but to measure ‘autophagic flux’ has driven the development of new or modified approaches to detecting autophile detection.

Biosensors for Monitoring Autophagy

This chapter highlights the application of biosensors used to monitor autophagic processes in live cells and focuses on the design and use of Rosella a dual wavelength emission biosensor based on a fusion of fluorescent proteins whose use exploits alterations in pH during autophagy.

How Shall I Eat Thee?

The terms being used to describe different types of lysosomal or vacuolar degradative pathways are considered, and each unique process needs a specific name to avoid confusion, and to eliminate the need for a lengthy description.

Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux

This work presents a luciferase-based reporter assay that measures autophagic flux in real time in living cells and demonstrates that this assay system is apt for the detection of dose- and stimulus-dependent differences in autophagy kinetics.

A novel method for autophagy detection in primary cells

The results indicate that healthy primary senescent CD8+ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age.

A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle

A new assay which is based on the fact that the number of autophagosomes is correlated with the amount of the LC3-II protein, which is specifically associated with Autophagosomal membranes is developed, suggested as being useful in the detection of autphagosomes in different phases of the cell cycle.

Methods for monitoring autophagy.

  • N. Mizushima
  • Biology, Chemistry
    The international journal of biochemistry & cell biology
  • 2004