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The phenotypic assessment of autophagy facilitates the establishment of workflows applicable to high throughput chemical drug screening campaigns by means of robotized automation platforms. Here, we describe a phenotypic screening routine for the identification of novel autophagic flux inducers from chemical libraries of small compounds. This screening routine encompasses fluorescent biosensor cells, automated liquid handling, robotized bioimaging as well as a multistep analysis pipeline. In this setting fluorescent biosensor cells expressing green fluorescent protein (GFP) conjugated to MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), as well as mCherry-GFP-LC3 tandem reporter cells are cocultured for the phenotypic assessment of LC3 puncta formation and lysosomal turnover. Image analysis and data processing facilitates the phenotypic separation of autophagic flux inducers from drugs that affect lysosomal acidification or fusion of lysosomes with the autophagosome.

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A Phenotypic Screening Routine for the Identification of Autophagic Flux Inducers

  • 章节

  • 发表日期：

    2025年

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  • Marion Leduc,
  • Sabrina Forveille,
  • Allan Sauvat,
  • Giulia Cerrato,
  • Guido Kroemer,
  • Oliver Kepp

摘要

The phenotypic assessment of autophagy facilitates the establishment of workflows applicable to high throughput chemical drug screening campaigns by means of robotized automation platforms. Here, we describe a phenotypic screening routine for the identification of novel autophagic flux inducers from chemical libraries of small compounds. This screening routine encompasses fluorescent biosensor cells, automated liquid handling, robotized bioimaging as well as a multistep analysis pipeline. In this setting fluorescent biosensor cells expressing green fluorescent protein (GFP) conjugated to MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), as well as mCherry-GFP-LC3 tandem reporter cells are cocultured for the phenotypic assessment of LC3 puncta formation and lysosomal turnover. Image analysis and data processing facilitates the phenotypic separation of autophagic flux inducers from drugs that affect lysosomal acidification or fusion of lysosomes with the autophagosome.