1. Electron microscopy
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Quantitative electron microscopy, immuno-TEM; monitor autophagosome number, volume
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2. Atg8/LC3 western blotting
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Western blot. The analysis is performed 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
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3. GFP-Atg8/LC3 lysosomal delivery and proteolysis
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Western blot ± lysosomal fusion or degradation inhibitors; the generation of free GFP indicates lysosomal/vacuolar delivery
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4. GFP-Atg8/LC3 fluorescence microscopy
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Fluorescence microscopy, FACS to monitor vacuolar/lysosomal localization. Also, increase in punctate GFP-Atg8/LC3 or Atg18/WIPI
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5. Tandem mRFP/mCherry-GFP fluorescence microscopy, Rosella
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Flux can be monitored as a decrease in green/red (yellow) fluorescence (phagophores, autophagosomes) and an increase in red fluorescence (autolysosomes)
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6. SQSTM1/p62 and related LC3 binding protein turnover
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The amount of SQSTM1 increases when autophagy is inhibited and decreases when autophagy is induced
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7. MTOR, AMPK and Atg1/ULK1 kinase activity
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Western blot, immunoprecipitation or kinase assays
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8. WIPI fluorescence microscopy
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Quantitative fluorescence analysis using endogenous WIPI proteins, or GFP- or Myc-tagged versions. Suitable for high-throughput imaging procedures.
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9. Transcriptional and translational regulation
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Northern blot, or qRT-PCR, autophagy-dedicated microarray
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10. Autophagic protein degradation
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Turnover of long-lived proteins to monitor flux
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11. Pex14-GFP, GFP-Atg8, Om45-GFP, mitoPho8Δ60
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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
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12. Autophagic sequestration assays
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Lysosomal accumulation by biochemical or multilabel fluorescence techniques, and TEM with a maturation inhibitor
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13. Turnover of autophagic compartments
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Electron microscopy with morphometry/stereology
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14. Autophagosome-lysosome colocalization and dequenching assay
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Fluorescence microscopy
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15. Sequestration and processing assays in plants
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Chimeric RFP fluorescence and processing, and light and electron microscopy
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16. Tissue fractionation
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Centrifugation, western blot and electron microscopy
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| 17. Degradation of endogenous lipofuscin |
Fluorescence microscopy |
