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Optimizing the Cell Painting assay for image-based profiling

Concannon, John, Aspesi Jr, Peter, Cimini, Beth, Chandrasekaran, Srinivas, Kost-Alimova, Maria, Miller, Lisa, Goodale, Amy, Fritchman, Briana, Byrne, Patrick, Garg, Sakshi, Jamali, Nasim, Logan, David, Lardeau, Charles-Hugues, Mouchet, Elizabeth, Singh, Shantanu, Shafqat Abbasi, Hamdah, Boyd, Justin, Gilbert, Tamara, Gnutt, David, Hariharan, Santosh, Hernandez, Desiree, Hormel, Gisela, Juhani, Karolina, Melanson, Michelle, Mervin, Lewis, Monteverde, Tiziana, Pilling, James, Skepner, Adam, Swalley, Susanne, Vrcic, Anita, Weisbart, Erin, Williams, Guy, Yu, Shan, Zapiec, Bolek and Carpenter, Anne (2023) Optimizing the Cell Painting assay for image-based profiling. Nature methods, 18. pp. 1981-2013. ISSN 1750-2799


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.

Item Type: Article
Keywords: Cell Painting, HCI, Image Based Profiling, Morphological Profiling
Date Deposited: 20 Jul 2023 00:45
Last Modified: 20 Jul 2023 00:45