Cell Painting, an image-based assay for morphological profiling

Abstract

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, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting 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 two weeks; feature extraction and data analysis take an additional 1-2 weeks.

Nature Inspired Small Molecules for Chemical Biology

“…However, advances in the culture and use of induced pluripotent stem cells and organoids are very promising steps in the right direction . For instance, the introduction of the high content phenotypic “cell‐painting assay” platform has provided a powerful tool to broadly assess biological compound profiles, provided that they induce changes in cellular morphology . Diverse fingerprints obtained from this assay can be used as a metric for the construction of focused chemical libraries that cover a broad range of biological space.…”

CellProfiler Analyst: interactive data exploration, analysis, and classification of large biological image sets

“…Its code base forms a solid foundation for integrating new classifiers into the tool, potentially including deep learning architectures. We also intend to integrate methods for constructing per-sample 'profiles' from raw morphological measurements to support morphological profiling applications (Caicedo et al, 2016;Bray et al, 2016).…”

Image‐Based Morphological Profiling Identifies a Lysosomotropic, Iron‐Sequestering Autophagy Inhibitor

“…Monitoring changes in cellular morphology induced by a hit molecule and comparing these to changes induced by a set of reference compounds with known modes of action and targets can provide target hypotheses. Morphological profiles can be extracted from simple brightfield images, and obtained from complex fluorescence‐based high‐content screens in which multiple subcellular compartments are labelled with various fluorophores . The multiplexed use of different fluorophores has been established in the “cell painting” assay, and has been proposed as a new strategy for determining whether a compound displays bioactivity in a very broad setting .…”

“…Morphological profiles can be extracted from simple brightfield images, and obtained from complex fluorescence‐based high‐content screens in which multiple subcellular compartments are labelled with various fluorophores . The multiplexed use of different fluorophores has been established in the “cell painting” assay, and has been proposed as a new strategy for determining whether a compound displays bioactivity in a very broad setting . In light of the promise and the potential of this approach, we explored the use of the cell painting assay for MMOA identification where other target identification methods had failed…”