Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles
Abstract 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...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-04-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-58765-8 |
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| author | Elena von Coburg Marlene Wedler Jose M. Muino Christopher Wolff Nils Körber Sebastian Dunst Shu Liu |
| author_facet | Elena von Coburg Marlene Wedler Jose M. Muino Christopher Wolff Nils Körber Sebastian Dunst Shu Liu |
| author_sort | Elena von Coburg |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-e0b958a5b5794f7ca16237395a4b0862 |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-e0b958a5b5794f7ca16237395a4b08622025-08-20T02:20:05ZengNature PortfolioNature Communications2041-17232025-04-0116112110.1038/s41467-025-58765-8Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cyclesElena von Coburg0Marlene Wedler1Jose M. Muino2Christopher Wolff3Nils Körber4Sebastian Dunst5Shu Liu6German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR)German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR)German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR)Screening Unit, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)Centre for Artificial Intelligence in Public Health Research, Robert Koch InstituteGerman Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR)German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR)Abstract 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.https://doi.org/10.1038/s41467-025-58765-8 |
| spellingShingle | Elena von Coburg Marlene Wedler Jose M. Muino Christopher Wolff Nils Körber Sebastian Dunst Shu Liu Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles Nature Communications |
| title | Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles |
| title_full | Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles |
| title_fullStr | Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles |
| title_full_unstemmed | Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles |
| title_short | Cell Painting PLUS: expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles |
| title_sort | cell painting plus expanding the multiplexing capacity of cell painting based phenotypic profiling using iterative staining elution cycles |
| url | https://doi.org/10.1038/s41467-025-58765-8 |
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