Imaging Autophagy. Issue 1 (1st July 2014)

• Record Type: Journal Article
• Authors: Karanasios, Eleftherios; Stapleton, Eloise; Manifava, Maria; Ktistakis, Nicholas T.
• Editors: Robinson, J. Paul; Darzynkiewicz, Zbigniew; Hoffman, Robert; Nolan, John P.; Rabinovitch, Peter S.; Watkins, Simon
• Is Part Of: Current protocols in cytometry. Volume 69:Issue 1(2014)
• Page Start: 12.34.1
• Page End: 12.34.16
• Publication Date: 2014-07-01
• Subjects: autophagy -- omegasomes -- ULK1 -- fluorescence microscopy -- live‐cell imaging; Cytology -- Laboratory manuals; Flow cytometry -- Laboratory manuals; Cell separation -- Laboratory manuals; Molecular biology -- Laboratory manuals; Flow Cytometry -- methods; Image Cytometry -- methods; Cell Separation -- methods; Cytological Techniques; Molecular Biology -- methods; Cell separation; Cytology; Flow cytometry; Molecular biology; Laboratory Manuals; Laboratory manuals; 571.6
• DOI: 10.1002/0471142956.cy1234s69
• Languages: English
• ISSNs: 1934-9297

Abstract

Abstract: Autophagy is a membrane‐trafficking pathway activated to deliver cytosolic material for degradation to lysosomes through a novel membrane compartment, the autophagosome. Fluorescence microscopy is the most common method used to visualize proteins inside cells, and it is widely used in the autophagy field. To distinguish it from the cellular background, the protein of interest (POI) is either fused with a genetically encoded fluorescent protein or stained with an antibody that is conjugated to an inorganic fluorescent compound. Genetic tagging of the POI allows its visualization in live cells, while immunostaining of the POI requires the fixation of cells and the permeabilization of cell membranes. Here we describe detailed protocols on how to visualize autophagy dynamics using fluorescence microscopy in live and fixed cells. We discuss the critical parameters of each technique, their advantages, and why the robustness is increased when they are used in tandem. Curr. Protoc. Cytom . 69:12.34.1‐12.34.16. © 2014 by John Wiley & Sons, Inc.