title: Methods for the Detection of Autophagy in Mammalian Cells.
abstract: Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Ziyan
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York.|Department of Medicine, Albert Einstein College of Medicine, Bronx, New York.|Diabetes Research Center, Albert Einstein College of Medicine, Bronx, New York.|Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Rajat
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Michael
authors.initials: M
authors.lastname: Aschner
mesh_terms: D000818:Animals; D000071182:Autophagosomes / Q000187:drug effects / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D054878:Lipoylation / Q000187:drug effects; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000071456:Sequestosome-1 Protein / Q000235:genetics / Q000378:metabolism; D023382:Toxicity Tests, Acute / Q000295:instrumentation / Q000379:methods; D000097573:Red Fluorescent Protein
chemical_list: D015415:Biomarkers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D000071456:Sequestosome-1 Protein; D000097573:Red Fluorescent Protein; C477012:MAP1LC3A protein, human
keywords: LC3; autophagic flux; autophagy; fluorescence microscopy; immunoblotting; p62