Methods for the detection of autophagy in mammalian cells

Research output: Contribution to journalArticle

14 Scopus citations

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 ofmultiple methods to accurately assess the autophagic activity in any given biological setting. C, 2016 by John Wiley & Sons, Inc.

Original languageEnglish (US)
Pages (from-to)1-26
Number of pages26
JournalCurrent Protocols in Toxicology
Volume2016
DOIs
StatePublished - Aug 2016

Keywords

  • Autophagic flux
  • Autophagy
  • Fluorescence microscopy
  • Immunoblotting
  • LC3
  • P62

ASJC Scopus subject areas

  • Toxicology

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