Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles.

Phenotypic changes in the morphology and internal organization of cells can indicate perturbations in cell functions. Therefore, imaging-based high-throughput phenotypic profiling (HTPP) applications such as Cell Painting (CP) play an important role in basic and translational research, drug discovery, and regulatory toxicology. Here we present the Cell Painting PLUS (CPP) assay, an efficient, robust and broadly applicable approach that further expands the versatility of available HTPP methods and offers additional options for addressing mode-of-action specific research questions. An iterative staining-elution cycle allows multiplexing of at least seven fluorescent dyes that label nine different subcellular compartments and organelles including the plasma membrane, actin cytoskeleton, cytoplasmic RNA, nucleoli, lysosomes, nuclear DNA, endoplasmic reticulum, mitochondria, and Golgi apparatus. In this way, CPP significantly expands the flexibility, customizability, and multiplexing capacity of the original CP method and, importantly, also improves the organelle-specificity and diversity of the phenotypic profiles due to the separate imaging and analysis of single dyes in individual channels.