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Flow Cytometric Analyses of Autophagic Activity using LC3-GFP fluorescence   

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Original research article

A brief version of this protocol appeared in:
Antimicrobial Agents and Chemotherapy
Jan 2013

Abstract

Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the LC3 punctae content might be. The method is based on the fact that intra-cellularly expressed LC3-GFP serves as a potential autophagic substrate for degradation. Therefore changes in total intracellular LC3-GFP fluorescence intensity is used as an indicator of cellular autophagic activity in living cells. Increased autophagic flux is expected to result in a progressive delivery of LC3-GFP to autolysosome where this substrate undergoes degradation. Therefore, enhanced autophagic flux is detected as a decreased total cellular GFP signal. On the other hand an inhibition of autophagic flux independent of the stage (autophagosome formation, maturation or acidification) leads to accumulation of undegraded LC3-GFP and may be detected as an enhanced intracellular GFP signal. (Caution: This methodology is based on the assumption that LC3-GFP is expressed constitutively by the model system. Data from analysis of substances or conditions influencing cellular LC3-GFP expression should be interpreted with care.)

Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Stankov, M., Panayotova-Dimitrova, D., Leverkus, M. and Behrens, G. (2014). Flow Cytometric Analyses of Autophagic Activity using LC3-GFP fluorescence. Bio-protocol 4(7): e1091. DOI: 10.21769/BioProtoc.1091.
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