PUB - Publikationen an der Universität Bielefeld

Cell Painting is an advanced imaging technique for drug discovery used to study cellular phenotypes by simultaneously labeling various organelles/structures and analyzing the resulting multidimensional phenotypic features through a sophisticated data analysis pipeline. Based on established phenotyping methodologies, this method has relied on incubation times of typically around 48 h for the assessment of phenotypic fingerprints. Here we provide evidence that earlier assessments show more robust results with increased significance of phenotypic fingerprints that better reflect primary physiological effects. Our study included compounds that range from representatives with modes of action that result in immediate phenotypic changes, such as energy metabolism inhibitors, to representatives that typically show pronounced phenotypes after several days, such as developmental inhibitors. Remarkably, we observed that for all compounds, primary cellular alterations were best detected at early timepoints after treatment, specifically at 6 h for Sf9 insect cells and shortly later timepoints for mammalian U2OS cells. Brief incubation periods enable the capture of primary effects of treatments while minimizing the influence of secondary changes as well as downstream phenotypic alterations like, for example, cell death. This enhances the specificity and accuracy of Cell Painting and consequently provides a more immediate depiction of primary actions from compounds. Notably, it also improves the efficiency of experimental workflows. In conclusion, we propose a more rapid assessment of cell phenotypes and morphology in the Cell Painting assay to enable a higher throughput in drug discovery screenings. Copyright © 2025. Published by Elsevier Inc.