{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T02:54:54Z","timestamp":1792810494718,"version":"4.5.0"},"publisher-location":"New York, NY","reference-count":22,"publisher":"Springer US","isbn-type":[{"value":"9781071649848","type":"print"},{"value":"9781071649855","type":"electronic"}],"license":[{"start":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T00:00:00Z","timestamp":1767225600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,1,2]],"date-time":"2026-01-02T00:00:00Z","timestamp":1767312000000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Cell Painting (CP) is a widely used imaging-based method for untargeted high-throughput phenotypic profiling (HTPP) of compounds or genetic perturbations that lead to morphological changes in cells. Here, we describe the Cell Painting PLUS (CPP) method, an efficient, robust, and broadly applicable HTPP approach that overcomes certain boundaries of the standard CP method. In CPP, an elution buffer enables iterative staining, elution, and re-staining of the same cells with at least seven fluorescent dyes labeling nine cellular compartments and organelles. In this way, CPP provides higher flexibility in selecting and combining various fluorescent dyes for customized multiplexing of phenotypic profiling screens. The separate imaging and analysis of single dyes in individual channels ensures a high specificity of the generated phenotypic profiles and thus enables precise insights into cellular processes and dysfunction. This chapter describes the process of generating CPP phenotypic profiling data that is based on automated staining and imaging procedures as well as a customized image analysis pipeline using the Harmony software. The resulting data can be further processed and visualized using customized KNIME software-based pipelines as described in von Coburg et al. (Nat Commun 16:3857, 2025) for unbiased and in-depth characterization of the modes-of-action of compounds or genetic perturbations in toxicology and biomedical research.<\/jats:p>","DOI":"10.1007\/978-1-0716-4985-5_2","type":"book-chapter","created":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T23:38:04Z","timestamp":1767310684000},"page":"31-50","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["The Cell Painting PLUS Method: Phenotypic Profiling Using Iterative Staining-Elution Cycles"],"prefix":"10.1007","author":[{"given":"Elena","family":"von Coburg","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sebastian","family":"Dunst","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,1,2]]},"reference":[{"issue":"1","key":"2_CR1","doi-asserted-by":"publisher","first-page":"3857","DOI":"10.1038\/s41467-025-58765-8","volume":"16","author":"E von Coburg","year":"2025","unstructured":"von Coburg E, Wedler M, Muino JM, Wolff C, Korber N, Dunst S, Liu S (2025) Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles. 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