(1102-C) High Content Imaging Is Now High Throughput: End-to-End High Content Imaging and Subcellular Analysis of Autophagy in Minutes
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Assays interrogating subcellular localization of fine puncta are vital to high content screening. However, imaging and analyzing this type of assay is a slow process, often taking a whole day or more after plates are prepared to get actionable data. The resolution required for examining such detail at scale means it takes several hours to scan a single plate, and quantitative spot-level analysis with subcellular localization may have to run overnight. This study details a high throughput, high content assay workflow for subcellular puncta quantification, measuring autophagic flux, taking minutes from plate to results using Araceli Endeavor™ and Araceli’s internal analysis tool. Autophagy assays are widely used, 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 aggregates; otherwise, it may fail to distinguish an increase in autophagic flux over the cell’s diffuse, homeostatic level of autophagy. In this experiment, we test several known modulators of autophagic flux in dilution, imaging them at submicron resolution with Araceli Endeavor®, with full well imaging in < 10minutes/plate. We demonstrate two distinct approaches to detecting autophagic vesicles, representative of two common staining strategies in high content: antibody staining, vesicle marker LC3b, and live cell dye, here a cationic amphiphilic dye selective binding autophagosomes. Overall, this poster demonstrates two end-to-end high content workflows with full well, whole plate imaging and analysis with sub-micron resolution and sub-cellular quantification in under 30 minutes.