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

Journal name:
Nature Protocols
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Published online


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.

At a glance


  1. The Cell Painting assay in U2OS and A549 cells.
    Figure 1: The Cell Painting assay in U2OS and A549 cells.

    The columns display the five channels imaged in the Cell Painting assay protocol (left to right) as imaged using the ImageXpress XLS microscope: Hoechst 33342 (DNA), concanavalin A (endoplasmic reticulum), SYTO 14 (nucleoli and cytoplasmic RNA), phalloidin (actin) and WGA (Golgi and plasma membrane), and MitoTracker Deep Red (mitochondria). Scale bars, 20 μm. See Table 1 for additional details about the stains and channels imaged.

  2. Overview of the strategy of morphological profiling using an image-based assay.
    Figure 2: Overview of the strategy of morphological profiling using an image-based assay.

    After perturbing, staining and imaging cells, the open-source software CellProfiler is used to extract ~1,500 morphological features of each cell. The collection of features is known as a profile: it reflects the phenotypic state of the cells in that sample, and it can be compared with other profiles to make inferences.

  3. Sample images from a small-molecule Cell Painting experiment using HUVEC cells.
    Figure 3: Sample images from a small-molecule Cell Painting experiment using HUVEC cells.

    Images are shown from a well treated with transfection reagent alone (negative control, top row) and an siRNA-treated well (bottom row), as imaged on an ImageXpress XLS microscope. The first five columns display the five channels imaged in the Cell Painting assay protocol, whereas the last column illustrates a merged image of the DNA stain (blue) and the nucleolar/cytoplasmic RNA stain (red), with the identified (i.e., segmented) nuclei and cell body outlines resulting from image analysis overlaid in white and magenta, respectively. Scale bars, 20 μm. See Table 1 for details about the stains and channels imaged. Each panel is 6% of a full image; for each well, nine images are acquired in a 3 × 3 grid, representing ~52% of the well area in total.


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Author information


  1. Imaging Platform, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Mark-Anthony Bray,
    • Shantanu Singh &
    • Anne E Carpenter
  2. Recursion Pharmaceuticals, Salt Lake City, Utah, USA.

    • Han Han,
    • Chadwick T Davis,
    • Blake Borgeson &
    • Christopher C Gibson
  3. Center for the Science of Therapeutics, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Cathy Hartland,
    • Maria Kost-Alimova &
    • Sigrun M Gustafsdottir


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.

Competing financial 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.

Corresponding authors

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Supplementary information

PDF files

  1. Supplementary Method (167 KB)

    Steps for producing per-well morphological profiles from single-cell measurements

  2. Supplementary Note (316 KB)

    Imaging considerations for wide-field versus confocal microscopes

Additional data