Application of cell painting techniques using an imaging flow cytometer 2417

Description Cell Painting (CP) is a method for staining organelles to establish a phenotype profile for each cell type and is used to identify normal cells from those responding to treatment. Fluorescent dyes are applied to paint cellular components, including DNA, mitochondria, and the cytoskeleton. Cells are treated with perturbagens, and images are analyzed for morphological changes. This allows for the collection of numerous morphological measurements on a single cell. Acquiring data for the CP assay is usually through High-Content Screening (HCS); however, HCS is typically applied to adherent cells in a plate-based assay. CP can be used for cells in suspension using the Cytek® Amnis® ImageStream®X Mk II system. In this study, we treated suspension cells with varying doses of the drug phenformin to determine if morphological changes could be detected and quantified. The CP assay was performed using dyes for nucleoli, mitochondria, ER, Golgi membrane, actin, and DNA to identify changes to cellular components. Image analysis software was used to establish the phenotype profile of the cells, and machine learning to determine which features had the greatest response and quantify any morphological changes. With the CP assay and the high-throughput performance of the Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer, we show how the mitochondria and other organelles aggregate and become granular when exposed to phenformin in suspension cells.