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