Optimizing the Cell Painting assay for image-based profiling

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations based on their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells, and predicting assay outcomes using machine learning, among many others. Here we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum, and mitochondria. The original protocol was updated in 2016 based on several years’ experience running it at two sites, after optimizing it by visual stain quality. Here we describe the work of the Joint Undertaking for Morphological Profiling (JUMP) Cell Painting Consortium, aiming to improve upon the assay via quantitative optimization, based on the measured ability of the assay to detect morphological phenotypes and group similar perturbations together. We find that the assay gives very robust outputs despite a variety of changes to the protocol and that two vendors’ dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1–2 weeks for a typically sized batch of 20 or fewer plates; feature extraction and data analysis take an additional 1–2 weeks. Key references using this protocol Virtual screening for small-molecule pathway regulators by image-profile matching () - recent work examining the ability to use collected Cell Painting profiles to screen for regulators of a number of diverse biological pathways. JUMP Cell Painting dataset: images and profiles from two billion cells perturbed by 140,000 chemical and genetic perturbations (DOI) - the description of the main JUMP master public data set, using this protocol in the production of >200 TB of image data and >200 TB of measured profiles. Key data used in this protocol Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes () - this paper provides the first step-by-step Cell Painting protocol ever released. ### Competing Interest Statement S.S. and A.E.C. serve as scientific advisors for companies that use image-based profiling and Cell Painting (AEC: Recursion, SS: Waypoint Bio, Dewpoint Therapeutics) and receive honoraria for occasional talks at pharmaceutical and biotechnology companies. D.G. is an employee of Bayer, AG, Pharmaceuticals. S.G., B.Z., G.H. are employees of Merck Healthcare KGaA, Darmstadt, Germany J.D.B. and T.G. were employed at Pfizer for the duration of this work. S.Y. was employed at Takeda for the duration of this work. S.E.S. was employed at Biogen for the duration of her contributions to this work. C.-H.L. was employed at Janssen Pharmaceutica at the time of writing. J.B.C & P.J.A. are employees of the Novartis Institutes for Biomedical Research, Cambridge MA, USA and declare no competing financial interests E.M., G.W., T.M, L.M. & J.P. are employees of AstraZeneca, Cambridge, UK K.J. was employed at AstraZeneca for the duration of this work. D.J.L. and S.H. are employees of Pfizer, Inc.