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 language | English (US) |
|---|---|
| Pages (from-to) | 1-26 |
| Number of pages | 26 |
| Journal | Current Protocols in Toxicology |
| Volume | 2016 |
| DOIs | |
| State | Published - Aug 1 2016 |
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Keywords
- Autophagic flux
- Autophagy
- Fluorescence microscopy
- Immunoblotting
- LC3
- P62
ASJC Scopus subject areas
- Toxicology
Cite this
Methods for the detection of autophagy in mammalian cells. / Zhang, Ziyan; Singh, Rajat; Aschner, Michael.
In: Current Protocols in Toxicology, Vol. 2016, 01.08.2016, p. 1-26.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Methods for the detection of autophagy in mammalian cells
AU - Zhang, Ziyan
AU - Singh, Rajat
AU - Aschner, Michael
PY - 2016/8/1
Y1 - 2016/8/1
N2 - 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.
AB - 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.
KW - Autophagic flux
KW - Autophagy
KW - Fluorescence microscopy
KW - Immunoblotting
KW - LC3
KW - P62
UR - http://www.scopus.com/inward/record.url?scp=85026634033&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85026634033&partnerID=8YFLogxK
U2 - 10.1002/cptx.11
DO - 10.1002/cptx.11
M3 - Article
C2 - 27479363
AN - SCOPUS:85026634033
VL - 2016
SP - 1
EP - 26
JO - Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.]
JF - Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.]
SN - 1934-9254
ER -