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Cell Painting is a high-throughput phenotypic profiling assay that uses fluorescent cytochemistry to visualize a variety of organelles and high-content imaging to derive a large number of morphological features at the single-cell level. Most Cell Painting studies have used the U-2 OS cell line for chemical or functional genomics screening. The Cell Painting assay can be used with many other human-derived cell types, given that the assay is based on the use of fluoroprobes that label organelles that are present in most (if not all) human cells. Questions remain, however, regarding the optimization steps required and overall ease of deployment of the Cell Painting assay to novel cell types. Here, we used the Cell Painting assay to characterize the phenotypic effects of 14 phenotypic reference chemicals in concentration–response screening mode across six biologically diverse human-derived cell lines (U-2 OS, MCF7 …