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Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes


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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Figure 1: The Cell Painting assay in U2OS and A549 cells.
Figure 2: Overview of the strategy of morphological profiling using an image-based assay.
Figure 3: Sample images from a small-molecule Cell Painting experiment using HUVEC cells.


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Research reported in this publication was supported in part by NIH R44 TR001197 (C.C.G.), NSF RIG DBI 1119830 (M.-A.B.), NIH R01 GM089652 (A.E.C.), and NSF CAREER DBI 1148823 (A.E.C.). The RNAi Cell Painting knockdown experiment used in this paper was previously published31 and was supported in part by the Slim Initiative for Genomic Medicine, a project funded by the Carlos Slim Foundation in Mexico. The authors thank the original developers of earlier versions of the protocol, who are the authors of the original paper describing the assay29; these authors are S.M. Gustafsdottir, V. Ljosa, K.L. Sokolnicki, J.A. Wilson, D. Walpita, M.M. Kemp, K. Petri Seiler, H.A. Carrel, T.R. Golub, S.L. Schreiber, P.A. Clemons, A.E. Carpenter, and A.F. Shamji. For enabling this work, Recursion Pharmaceuticals thanks the University of Utah Core Facilities (J. Phillips), specifically the Drug Discovery Core (B. Luo) and the Fluorescent Imaging Core (C. Rodesch). We thank members of the Broad Institute's Center for the Development of Therapeutics, especially T.P. Hasaka, for technical assistance. We also thank A. Kozol for proofreading the Equipment and Reagents sections and for testing the image analysis workflow, and A. Berger and X. Wu for offering helpful comments and suggestions during manuscript preparation.

Author information




All authors contributed to writing this protocol. C.T.D., H.H., and S.M.G. contributed to describing the benchwork aspects of the protocol. C.H., M.K.-A., M.-A.B., and C.T.D. contributed to updates to the experimental design. M.-A.B. and S.S. contributed most heavily to describing the computational aspects of the protocol. A.E.C., C.C.G., B.B., and S.S. contributed most heavily to describing the rationale and the experimental design.

Corresponding authors

Correspondence to Christopher C Gibson or Anne E Carpenter.

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Competing interests

Recursion Pharmaceuticals is a biotechnology company in which C.C.G., B.B., C.T.D., H.H., and A.E.C. have real or optional ownership interest.

Supplementary information

Supplementary Method

Steps for producing per-well morphological profiles from single-cell measurements (PDF 163 kb)

Supplementary Note

Imaging considerations for wide-field versus confocal microscopes (PDF 308 kb)

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Bray, MA., Singh, S., Han, H. et al. Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes. Nat Protoc 11, 1757–1774 (2016).

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