Abstract 2069: High content imaging without sacrificing coverage, resolution or speed: Functional validation through multiple assays

Abstract Traditionally, high content screening (HCS) required compromise: sacrificing resolution, sample coverage or speed. This has limited high content screening’s utility as a high throughput tool, as speed is essential to cancer research at scale. Using a new approach, Araceli Endeavor® offers a solution to this compromise. This system images 96, 384 or 1536 -well high content microplates in under 10 minutes with full well coverage and sub-micron resolution, enabling high content screening at high throughput speeds without compromising resolution or coverage. Paired with the Clairvoyance™ analysis software, plates are analyzed at similar speeds, going from images to object-level data in 5-25 minutes. As with any new technology, validation is needed. Here we validate Endeavor and Clairvoyance at the level of the bioassay, using a range of common high content assays across different modalities. Assays are chosen to quantifiably demonstrate sensitivity, breadth, and broad applicability. Performance was benchmarked by using well-characterized compounds, matching published EC50/IC50 values when possible. We examined submicron-level nuclear spot counting, measuring histone yH2Ax and micronuclei formation after genotoxic insult. Intensity-based assays include cellular positivity, here live/dead, and subcellular translocation with transcription factor NFΚB after induction with cytokines Il1b and TNFα. Looking at cytosolic vesicle aggregation, we measured autophagic flux with two distinct modalities, comparative imaging and analysis of live cell dye and immunohistochemistry-based detection yielded consistent EC50 values between methods. Overall, these assays yield consistent, robust results corresponding to literature values, showing the importance of full well coverage and resolution. We demonstrate how assays that formerly took hours can be effectively imaged and analyzed without sacrificing quality in 10-30 minutes. Citation Format: Matthew Boisvert, Josh Kieler, Dan DiSepio, Don Weldon. High content imaging without sacrificing coverage, resolution or speed: Functional validation through multiple assays abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2069.