(1076-A) High content imaging is now high throughput: End to end high content imaging and analysis of autophagy on a subcellular level in minutes

Abstract: Assays interrogating subcellular localization of fine puncta are vital to high content screening. However, the time required for imaging and analysis is a slow process, taking a whole day after plates are prepared to generate results. The resolution required for examining such detail at scale means it takes several hours to scan a single plate, and quantitative spot-level localization analysis may have to run overnight. This study details a high throughput, high content assay workflow, taking minutes from plate to results using Araceli Endeavor™ and Araceli Voyager™. Measuring autophagic flux is a paradigmatic example of a spot-level high content assay, where the autophagosome marker LC3b identifies and quantifies the response to cellular damage and stress. Autophagic flux is a widely used assay particularly in the fields of aging and neurodegenerative research, and several FDA-approved cancer treatments specifically target autophagy. This assay requires sufficient detection to resolve and quantify individual LC3b aggregates; otherwise, it may fail to distinguish an increase in autophagic flux over the cell’s diffuse, homeostatic level of LC3b. In this experiment, we test several known inducers of autophagic flux in dilution, imaging them at submicron resolution with Araceli Endeavor™, with full well imaging in < 10minutes/plate. We then analyzed these data through 2 pipelines: showing that CellProfiler, an open-source image analysis package, and Araceli Voyager™, a recently debuted high throughput analysis software package, produce equivalent results, accurately detecting autophagosomes in with compounds with high Z’ scores, indicative of a robust effect with minimal variability. Notably, analysis times differed significantly between systems: CellProfiler required 2 full days of assay refinement plus 4-5 hours processing time per plate, for analysis Voyager accomplished in 30 minutes/plate processing, with only 20 minutes needed to refine the assay parameters. This functions as a proof of concept for the Araceli platform as a bona fide high throughput high content system, showcasing an accurate and efficient methodology broadly applicable to aggregate and spot quantification of all kinds.