High Content Is Now High Throughput: End-to-End High Content Imaging and Subcellular Analysis of Autophagy in Minutes

Authors: Matthew Boisvert and Josh Kieler

Introduction: Measuring Autophagic Flux at High Throughput Speeds

  • Cells eliminate intracellular waste and defective components through autophagy (“self-eating”)
  • A key therapeutic target for aging-related dysfunction, autophagy is implicated in neurodegeneration and cancer
  • Autophagic flux can be measured by quantifying autophagic vesicles, visualized through examining marker protein LC3b or dyes activated by vesicle-specific conditions
  • High content imaging (HCI) allows for cell-level resolution, visualizing aggregates and defining cell borders, quantifying spots per cell
  • Resolving aggregated autophagosomes is essential, cell lines often have baseline levels of autophagy, generally non-aggregated
  • Screening large compound libraries for autophagic flux requires speed, low variability and accurate detection
  • Using the Araceli Endeavor® HCI Platform this assay demonstrates:
    • Assay validation with four mechanistically different compounds
    • Submicron resolution to accurately visualize aggregates
    • Elimination of variability with whole well imaging
    • <10-minute scan times for 96, 384 and 1536-well high content plates
    • Flexibility: equivalent results with immunohistochemistry (IHC) and cell dye-based workflows

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