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Nat Protoc DOI:10.1038/nprot.2016.105

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

Publication TypeJournal Article
Year of Publication2016
AuthorsBray, M-A, Singh, S, Han, H, Davis, CT, Borgeson, B, Hartland, C, Kost-Alimova, M, Gustafsdottir, SM, Gibson, CC, Carpenter, AE
JournalNat Protoc
Date Published2016 09
KeywordsCell Line, Tumor, Cell Shape, Cell Size, Fluorescent Dyes, Humans, Image Processing, Computer-Assisted, Molecular Imaging, Staining and Labeling

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.


Alternate JournalNat Protoc
PubMed ID27560178
PubMed Central IDPMC5223290
Grant ListR01 GM089652 / GM / NIGMS NIH HHS / United States
R44 TR001197 / TR / NCATS NIH HHS / United States