Abstract 2063: Using the edge wells in 96-well plates without increasing variability in cytotoxicity assays through thermal controls

Abstract

Abstract There are very few things as fundamental to cancer research as the 96-well-plate, yet edge effect has continued to cause problems since its introduction. The most common remedy for edge effect is to avoid using edge wells altogether, which eliminates 37% of the plate from the assay. If all of those wells could be used, an estimated USD $119M could be saved in research costs annually across the globe. We have shown previously that maintaining constant thermal control of plating conditions reduces variability in stem cell toxicity assays. Here, we ask the question: can we use the edge wells in cancer screening assays if we control thermal changes that happen in the traditional lab as cells are plated at room temperature and incubated at 37 degrees C? We had the null hypothesis that including the edge wells would add variability to an A549 cytotoxicity assay, even if the cell handling and incubation temperatures were matched and held constant. We plated A549 cells at either room temperature (like a BSC) or at 37 degrees C using the Xvivo System, which maintains constant temperature, oxygen, CO2, and relative humidity levels around cell handling operations. The cells were treated with serial dilutions of peracetic acid/hydrogen peroxide solution. After incubation for 3 days, the plates were stained with Crystal Violet and assessed for cell density (n=3 separate experiments). The null hypothesis was disproven. We found that if the plates were maintained at 37 degrees C during cell handling as well as incubation, that cell distribution was random and even across all wells, even edge wells. We found that there was no statistical difference between edge well and center well results in cytotoxicity assays. Edge wells were usable without increasing variability in the assays. This is consistent with earlier findings showing that when temperature changes between cell plating and incubation were averted, stem cell distribution across the plate was random and the edge effect was eliminated. Citation Format: Alicia D. Henn, Shannon Darou, Randy Yerden. Using the edge wells in 96-well plates without increasing variability in cytotoxicity assays through thermal controls [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(6_Suppl):Abstract nr 2063.