Autophagic flux was determined by immunoblot analysis of cells treated with or without with 20 mM NH4Cl and 100 μM leupeptin to block lysosomal proteolysis1 (link). The difference in LC3 levels between cells treated with or without the inhibitors was used to calculate autophagic flux, whereas the difference in LC3 levels at two times during the inhibition of proteolysis was used as an index of autophagosome formation. Autophagic flux was also measured upon transfection of the cells with the tandem reporter mCherry-GFP-LC3 (Addgene38 (link)). Cells were imaged 24 hours after transfection and the number of mCherry positive vesicles (autophagic vacuoles), mCherry and GFP positive vesicles (autophagosomes), and mCherry-only positive vesicles (autolysosomes) was calculated after thresholding, using the particle measure tool of Image J software (NIH).
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Quantifying Autophagic Flux and Dynamics
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Autolysosomes
Autophagic
Autophagosomes
Block
Cells
Immunoblot analysis
Inhibition
Inhibitors
Leupeptin
Lysosomal
Proteolysis
Transfection
Corresponding Organization : Albert Einstein College of Medicine
Other organizations : Cornell University
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Variable analysis
independent variables
- Treatment with 20 mM NH4Cl and 100 μM leupeptin to block lysosomal proteolysis
- Transfection of cells with the tandem reporter mCherry-GFP-LC3
- Autophagic flux determined by immunoblot analysis
- Difference in LC3 levels between cells treated with or without the inhibitors
- Difference in LC3 levels at two times during the inhibition of proteolysis
- Number of mCherry positive vesicles (autophagic vacuoles), mCherry and GFP positive vesicles (autophagosomes), and mCherry-only positive vesicles (autolysosomes) calculated after thresholding, using the particle measure tool of Image J software
- Not explicitly mentioned
- Not mentioned
- Not mentioned
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