Title: Methods for the Detection of Autophagy in Mammalian Cells.
Authors: Zhang, Ziyan; Singh, Rajat; Aschner, Michael
Published In Curr Protoc Toxicol, (2016 08 01)
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.
PubMed ID: 27479363
MeSH Terms: Animals; Autophagosomes/drug effects; Autophagosomes/metabolism*; Autophagy*/drug effects; Biomarkers/metabolism; Cell Line; Cells, Cultured; Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; Humans; Immunoblotting; Lipoylation/drug effects; Luminescent Proteins/genetics; Luminescent Proteins/metabolism; Mammals; Microscopy, Fluorescence; Microtubule-Associated Proteins/genetics; Microtubule-Associated Proteins/metabolism; Phosphatidylethanolamines/metabolism; Protein Processing, Post-Translational/drug effects; Protein Transport/drug effects; Recombinant Fusion Proteins/metabolism; Sequestosome-1 Protein/genetics; Sequestosome-1 Protein/metabolism; Toxicity Tests, Acute/instrumentation; Toxicity Tests, Acute/methods