Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes
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Abstract
In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, which is a morphological profiling assay that multiplexes six fluorescent dyes, imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multiwell plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Next, an automated image analysis software identifies individual cells and measures ∼1,500 morphological features (various measures of size, shape, texture, intensity, and so on) to produce a rich profile that is suitable for the detection of subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes 2 weeks; feature extraction and data analysis take an additional 1-2 weeks.
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The concept of phenotypic profiling was pioneered by the laboratory of A.E. Carpenter at the Broad Institute of Harvard and MIT and has been successfully implemented, optimised, and standardised for high-throughput phenotypic profiling at various sites worldwide.
“The concept of PP was pioneered by the laboratory of A.E. Carpenter at the Broad Institute of Harvard and MIT (Gustafsdottir et al. 2013; Bray et al. 2016) and has quickly been successfully implemented, optimised, and standardised for high-throughput phenotypic profiling at various sites worldwide (Cimini et al. 2023; Tromans-Coia et al. 2023).”
From Bridging imaging-based in vitro methods from biomedical research to regulatory toxicology (2025) · cites this paper as Bray et al. 2016
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Experimental — accuracy not yet measuredRxRx1-wilds images were acquired via fluorescent microscopy using a 6-channel variant of the Cell Painting assay.
“These were acquired via fluorescent microscopy using a 6-channel variant of the Cell Painting assay (Bray et al., 2016).”
From WILDS: A Benchmark of in-the-Wild Distribution Shifts (2020) · cites this paper as Bray et al., 2016
The Introduction (Development of the protocol; page unavailable) identifies Cell Painting as a fluorescence microscopy assay, so the citation supports the assay being named. It describes six stains imaged in five channels, however, and does not establish that RxRx1-wilds images used a six-channel variant. (LLM, conf=0.86)
Experimental — accuracy not yet measured
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- [39]Bray M-A Carpenter A Assay Guidance Manual Sittampalam GS Eli Lilly & Company and the National Center for Advancing Translational Sciences 2013 22553861
- [40]Iversen PW Assay Guidance Manual Sittampalam GS Eli Lilly & Company and the National Center for Advancing Translational Sciences 2012 22553861
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- Authors
- Bray, M.-A., Singh, S., Han, H., Davis, C. T., Borgeson, B., Hartland, C., Kost-Alimova, M., Gustafsdottir, S. M., Gibson, C. C., & Carpenter, A. E.
- Affiliations
- Broad Institute (US)
- Recursion (United States) (US)
- Corresponding author
- Gibson, Christopher C
- Carpenter, Anne E
- Venue
- Nature Protocols
- Published
- 2016-08-25
- Volume
- 11
- Issue
- 9
- Pages
- 1757-1774
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC5223290
- OpenAlex
- W2498090354
- Cited by
- 1,090
- References
- 47
- Citations by year
- 2026: 180 · 2025: 205 · 2024: 191 · 2023: 143 · 2022: 93 · 2021: 98 · 2020: 81 · 2019: 44 · 2018: 30 · 2017: 20 · 2016: 4 · 2012: 1
- FWCI
- 20.62
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Nature Protocols (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Science Foundation (1119830, 1148823, CAREER)
- University of Utah
- Broad Institute
- National Institutes of Health (NIH-R01, R44TR001197, GM089652, NIH R01 GM089652)
- National Institute of General Medical Sciences (R01GM089652)
- National Center for Advancing Translational Sciences
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Physical Sciences › Computer Science › Computer Vision and Pattern Recognition › Digital Imaging for Blood Diseases
- Keywords
- Cell Painting, morphological profiling, high-content imaging, cell morphology, automated image analysis, chemical perturbation, gene perturbation, disease signatures, high-throughput imaging
- MeSH terms
- Fluorescent Dyes, Humans, Image Processing, Computer-Assisted, Staining and Labeling, Cell Line, Tumor, Cell Size, Cell Shape, Molecular Imaging
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