Table 4.

Recommended methods for monitoring autophagy.

Method		Description
1.	Electron microscopy	Quantitative electron microscopy, immuno-TEM; monitor autophagosome number, volume, and content/cargo.
2.	Atg8/LC3 western blotting	Western blot. The analysis is carried out in the absence and presence of lysosomal protease or fusion inhibitors to monitor flux; an increase in the LC3-II amount in the presence of the inhibitor is usually indicative of flux.
3.	GFP-Atg8/LC3 lysosomal delivery and proteolysis	Western blot +/− lysosomal fusion or degradation inhibitors; the generation of free GFP indicates lysosomal/vacuolar delivery.
4.	GFP-Atg8/LC3 fluorescence microscopy	Fluorescence microscopy, flow cytometry to monitor vacuolar/lysosomal localization. Also, increase in punctate GFP-Atg8/LC3 or Atg18/WIPI, and live time-lapse fluorescence microscopy to track the dynamics of GFP-Atg8/LC3-positive structures.
5.	Tandem mRFP/mCherry-GFP fluorescence microscopy, Rosella	Flux can be monitored as a decrease in green/red (yellow) fluorescence (phagophores, autophagosomes) and an increase in red fluorescence (autolysosomes).
6.	Autophagosome quantification	FACS/flow cytometry.
7.	SQSTM1 and related LC3 binding protein turnover	The amount of SQSTM1 increases when autophagy is inhibited and decreases when autophagy is induced, but the potential impact of transcriptional/translational regulation or the formation of insoluble aggregates should be addressed in individual experimental systems.
8.	MTOR, AMPK and Atg1/ULK1 kinase activity	Western blot, immunoprecipitation or kinase assays.
9.	WIPI fluorescence microscopy	Quantitative fluorescence analysis using endogenous WIPI proteins, or GFP- or MYC-tagged versions. Suitable for high-throughput imaging procedures.
10.	Bimolecular fluorescence complementation	Can be used to monitor protein-protein interaction in vivo.
11.	FRET	Interaction of LC3 with gangliosides to monitor autophagosome formation.
12.	Transcriptional and translational regulation	Northern blot, or qRT-PCR, autophagy-dedicated microarray.
13.	Autophagic protein degradation	Turnover of long-lived proteins to monitor flux.
14.	Pex14-GFP, GFP-Atg8, Om45-GFP, mitoPho8Δ60	A range of assays can be used to monitor selective types of autophagy. These typically involve proteolytic maturation of a resident enzyme or degradation of a chimera, which can be followed enzymatically or by western blot.
15.	Autophagic sequestration assays	Accumulation of cargo in autophagic compartments in the presence of lysosomal protease or fusion inhibitors by biochemical or multilabel fluorescence techniques.
16.	Turnover of autophagic compartments	Electron microscopy with morphometry/stereology at different time points.
17.	Autophagosome-lysosome colocalization and dequenching assay	Fluorescence microscopy.
18.	Sequestration and processing assays in plants	Chimeric RFP fluorescence and processing, and light and electron microscopy.
19.	Tissue fractionation	Centrifugation, western blot and electron microscopy.
20.	Degradation of endogenous lipofuscin	Fluorescence microscopy.