Abstract
We present a 3D assay for the quantification of the autophagic flux in live cell spheroids by using the fluorescent reporter mRFP-GFP-LC3. The protocol describes the formation of the spheroids from the astrocytoma cell line U343, live long-term 3D fluorescence imaging of drug-treated spheroids, and the image processing workflow required to extract quantitative data on the autophagic flux.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Autophagy / drug effects*
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Cell Line, Tumor
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Cell Proliferation
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Cell Survival
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Gossypol / pharmacology
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Green Fluorescent Proteins / metabolism
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Humans
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Luminescent Proteins / metabolism
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Microscopy, Confocal
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Microscopy, Fluorescence / methods*
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Molecular Imaging / methods*
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Red Fluorescent Protein
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Sirolimus / pharmacology
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Spheroids, Cellular / cytology*
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Spheroids, Cellular / drug effects*
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Spheroids, Cellular / metabolism
Substances
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Luminescent Proteins
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Green Fluorescent Proteins
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Gossypol
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Sirolimus