What is Cell Painting?
Cell Painting is a high-content, multiplexed image-based assay used for cytological profiling. In a Cell Painting assay, up to six fluorescent dyes are used to label different components of the cell including the nucleus, endoplasmic reticulum, mitochondria, cytoskeleton, Golgi apparatus, and RNA. The goal is to “paint” as much of the cell as possible to capture a representative image of the whole cell.
Automated image analysis software is used to extract feature measurements from each cell. The number of unique measurements is usually in the range of 100 to 1000 per cell. These measurements typically include intensity, texture, shape, size as well as the proximity of an object to its neighboring structure, which provides an indication of the spatial relationship between organelles. Together, these measurements form the phenotypic profile.

Nucleus
Dye: Hoechst 33342

Mitochondria
Dye: MitoTracker Deep Red

Endoplasmic reticulum
Dye: Concanavalin A/Alexa Fluor 488 conjugate

Nucleoli, cytoplasmic RNA
Dye: SYT0 14 green fluorescent nucleic acid stain

F-actin cytoskeleton, Golgi, plasma membrane
Dye: Phalloidin/Alexa Fluor 568 conjugate, wheat-germ agglutinin/Alexa Fluor 555 conjugate

Composite image consisting of Red: Actin and golgi; Green: ER; and Blue: nuclei
Cell Painting for phenotypic profiling
The phenotypic profile of a cell reveals the specific biological state of a cell. More specifically, it can be used to interrogate biological perturbations because the cellular morphology is influenced by factors such as metabolism, genetic and epigenetic state of the cell, and environmental cues. In addition, it can be used to characterize healthy cells from diseased cells. Because a phenotypic profile is an aggregation of a large number of measurements, it is more sensitive to deviations or changes to those features extracted from Cell Painting assay. In other words, a phenotypic profile can capture certain characteristics of the cells that may not be obvious to the naked eye.
In an interview with the Science Explorer, Angeline Lim, PhD., explained, “I like to think of Cell Painting or phenotypic profiling as analogous to facial recognition. It's a little bit like how Facebook or iPhoto tags people. Once you have a photograph and you tag it, the software in the background extracts information about the photo and calculates a profile. When another photo pops up, the software compares its profile to that of the previous photo to see if they are the same or different. This is basically how Cell Painting works. With Cell Painting, we expect the image analysis part to cluster like cells from unlike cells, or diseased cells from healthy cells. This potentially has many applications, especially in drug discovery.”
Examples of treated cells and phenotypic changes
Here is an example in which treated cells show obvious changes in their phenotypes—only three of the five wavelengths are shown in this composite: nuclei in blue, endoplasmic reticulum in green, actin & Golgi in red.

Control, untreated cells.

Cells treated with rotenone, a toxin frequently used in insecticides. It is also known to inhibit mitochondrial ATP production and has been shown to have anticancer activity in various cancer cells.

Cells treated with chloroquine. Chloroquine was first developed for malaria treatment.
General workflow for Cell Painting assay
One of the advantages in the Cell Painting assay is that the overall workflow is one that is familiar to many biologists. First, you plate the cells. Next, you introduce a type of perturbation; it can be chemical or genetic (e.g., adding of compounds, small molecules, or an RNAi library). After a suitable incubation period, the cells are stained with a set of Cell Painting dyes. It is also possible to use other dye combinations that are more suited to your specific assay.
Once the cells are “painted,” cell images are acquired with a high-content imager such as our ImageXpress® Confocal HT.ai, the latest addition to our portfolio of high-content imaging system. An automated image analysis software like our MetaXpress® or our IN Carta image analysis software is used for feature extraction where cells and their components are identified and measured. Finally, the measurements are further processed using various data analysis tools to create and compare phenotypic profiles, perform clustering analysis, and identify targets to derive the morphological profiles.
- Plate cells into labware (384-well plate)
- Treat cells with chemical or genetic perturbation (e.g., RNAi, CRISPR,/Cas9) or viruses.
- Stain with fluorescent dyes (e.g., Hoechst, Phalloidin, MitoTracker)
- Acquire cell images with high-content imaging system
- Analyze cell images to extract features and measurements using automated image analysis software
- Derive morphological profiles from measurements
Learn more about Cell Painting
Cell Painting is emerging as a valuable tool with its many important potential applications particularly in drug discovery. Learn how to optimize high-content imaging capabilities and perform multiplexed assays using preconfigured or custom modules from MetaXpress software to provide a data rich, phenotypic profile.
Resources for Cell Painting
Publications
Overcoming ‘Analysis Paralysis’ in Cell Painting With Artificial Intelligence
Overcoming ‘Analysis Paralysis’ in Cell Painting With Artificial Intelligence
Gone are the days of only measuring single parameters in cell-based experiments. Instead, researchers should widen their focus, namely with the help of innovations like the Cell Painting ass…
Scientific Poster
Image-based phenotypic profiling using Cell Painting in a 3D breast cancer spheroid model
Image-based phenotypic profiling using Cell Painting in a 3D breast cancer spheroid model
Most potential oncology drugs fail the drug development pipeline, despite having promising data for their efficacy in vitro. This further incentivize the need for identifying in vitro models…
Blog
Advanced technology for automated 3D biology workflows #SLASEurope2022
Advanced technology for automated 3D biology workflows #SLASEurope2022
SLAS Europe 2022, hosted numerous sessions packed with the latest research on emerging topics as well as sessions and panel discussions focused on how to build, and succeed, in a…
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AI-Powered Phenotypic Profiling Revolutionizes Drug Discovery
AI-Powered Phenotypic Profiling Revolutionizes Drug Discovery
Today, nine out of ten drugs fail in clinical trials. Furthermore, it takes over a decade and an average cost of $2 billion to develop and approve each medicine.1 An important underlying rea…
Publications
A Picture Paints a Thousand… New Therapies?
A Picture Paints a Thousand… New Therapies?
Cell painting is a high-content profiling technology that uses up to six fluorescent dyes to visualize specific cellular components at the single-cell level. The assay essentially “paints” i…
Brochure
StratoMineR
StratoMineR
Core Life Analytics’ StratoMineR™ software helps biologists analyze the complex data derived from high-content image analysis. A powerful, intuitive workflow allows users to port data analyz…
Application Note
3D Immune Cell Migration
3D Immune Cell Migration
Cell migration is an essential process for many biological phenomena such as early embryonic development and immune cell response. During inflammation, the recruitment of T cells through…
Scientific Poster
Phenotypic profiling based on the Cell Painting assay
Phenotypic profiling based on the Cell Painting assay
Multiparametric high-content screening approaches, such as the Cell Painting assay, are increasingly being used in many applications ranging from drug discovery programs to functional genomi…
Application Note
Label-free cell segmentation with IN Carta SINAP application module
Label-free cell segmentation with IN Carta SINAP application module
Label-free cell analysis provides a better alternative to using fluorescent dyes as it enables scientists to image live cells under near-native conditions – examining biological processes…
Blog
Engineering Next-gen Organoids with Automated Lab Workflows at #SLAS2022
Engineering Next-gen Organoids with Automated Lab Workflows at #SLAS2022
SLAS2022, the Society for Lab Automation and Screening conference offered another exciting year for learning about innovative laboratory technologies. Whether you attended in-person…
Application Note
Improving the robustness of Cell Painting with a near-infrared label and advanced image and data analytics
Improving the robustness of Cell Painting with a near-infrared label and advanced image and data analytics
Image-based phenotypic profiling approaches such as the widely used Cell Painting assay use high-content imaging along with multiparametric readouts to study biological, genetic, and…
Scientific Poster
Automation and high content imaging of 3D triple-negative breast cancer patient-derived
Automation and high content imaging of 3D triple-negative breast cancer patient-derived
Triple negative breast cancer is a clinically aggressive tumor subtype, with high rates of metastasis, recurrence, and drug resistance. Currently there are no clinically approved small molec…
Scientific Poster
Improving the robustness of Cell Painting with a near-infrared label and advanced image and data analytics
Improving the robustness of Cell Painting with a near-infrared label and advanced image and data analytics
Image-based phenotypic profiling approaches, such as the widely-used Cell Painting assay, use high-content imaging along with multiparametric readouts to study biological, genetic, and chemi…
Blog
Stem cell science insights and breakthroughs presented at #ISSCR2021
Stem cell science insights and breakthroughs presented at #ISSCR2021
If you didn't get a chance to visit us at our poster sessions during ISSCR 2021, don’t fret. We've gathered all our sessions right here for you. The ISSCR Annual Meeting brought…
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Organoids for disease modeling and in vitro drug screening
Organoids for disease modeling and in vitro drug screening
3D cell models representing various tissues were successfully used for studying complex biological effects, tissue architecture, and functionality. However, the complexity of 3D models remai…
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Monitoring organoid development and characterization of calcium oscillation activities in iPSC-derived 3D cerebral organoids
Monitoring organoid development and characterization of calcium oscillation activities in iPSC-derived 3D cerebral organoids
Cerebral organoids are a rapidly developing technology that has great potential for understanding brain development and neuronal diseases. They can also be used for testing effects of compou…
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High-throughput assessment of compound-induced pro-arrhythmic effects in human IPSC-derived cardiomyocytes
High-throughput assessment of compound-induced pro-arrhythmic effects in human IPSC-derived cardiomyocytes
Development of biologically relevant and predictive cell-based assays for compound screening and toxicity assessment is a major challenge in drug discovery. The focus of this study was to es…
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Deep learning-based image analysis for label-free live monitoring of iPSC 3D organoid cultures
Deep learning-based image analysis for label-free live monitoring of iPSC 3D organoid cultures
Complex 3D biological models such as organoids and patient derived spheroids are gaining popularity in many biomedical research areas because they more closely recapitulate the in vivo tissu…
Publications
Project Profile: Organoid Innovation Center
Project Profile: Organoid Innovation Center
Lab Manager speaks to Dan O’Connor, vice president, drug discovery, Molecular Devices, about the company’s Organoid Innovation Center in San Jose, CA. The center is 180 sq. ft., within a lar…
Blog
Tips for running a successful live cell imaging experiment
Tips for running a successful live cell imaging experiment
There have been significant advancements in microscopy and camera technology, as well as advancements in technologies for labeling molecules of interest over the past decade. These…
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Shaping the Future of Organoid Research
Shaping the Future of Organoid Research
Molecular Devices, a provider of innovative life science technology, recently unveiled a brand new, first-of-its-kind Organoid Innovation Center. Situated at the company’s global headquarter…
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Overcome the challenges of high-content cell analysis through AI/machine learning
Overcome the challenges of high-content cell analysis through AI/machine learning
Artificial intelligence (AI) is finding its way into many aspects of modern life, from autonomous vehicles to voice-powered personal assistants, and even the creation of art. But it…
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Automated 3D cell-based assays using a novel flow chip system and high-content imaging
Automated 3D cell-based assays using a novel flow chip system and high-content imaging
There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translation research. Significant progress has been made…
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Novel assay methods for cancer patient derived organoids
Novel assay methods for cancer patient derived organoids
In recent years, researchers have transitioned from traditional 2D assays to more complex 3D cell models, as they are shown to recapitulate the in vivo environment and serve as a more predic…
Blog
How Cell Painting is making its mark on drug discovery
How Cell Painting is making its mark on drug discovery
Have you ever heard the old adage, “A picture is worth a thousand words?” When it comes to Cell Painting, this saying is especially true. Cell Painting is a high-content,…
Publications
Danaher’s SCIEX and Molecular Devices Businesses Debut New Products
Danaher’s SCIEX and Molecular Devices Businesses Debut New Products
It has been a busy beginning of the year for Danaher’s Life Sciences segment. In January, the business reported 2020 revenues of $10.6 billion, representing 47% of the total revenues of scie…
Scientific Poster
Using a laser light source for high-content automated imaging
Using a laser light source for high-content automated imaging
In this study, we demonstrate improvement in assay sensitivity, precision, and speed of acquisition with a new configuration of the ImageXpress® Confocal HT.ai High-Content Imaging System th…
Scientific Poster
Simplified workflow for phenotypic profiling based on the Cell Painting assay
Simplified workflow for phenotypic profiling based on the Cell Painting assay
Multiparametric high-content screening approaches, such as the Cell Painting assay, are increasingly being used in many applications ranging from drug discovery programs to functional genomi…
Scientific Poster
Lung organoids for disease modeling and toxicity assessment by 3D high-content imaging and analysis
Lung organoids for disease modeling and toxicity assessment by 3D high-content imaging and analysis
Organoid models have increasingly gained popularity in biologic research and screening to recapitulate complexity of real tissues. To model the in vivo human lungs, we have cultured primary…
Application Note
Improve sensitivity, speed, and assay quality for complex biological assays
Improve sensitivity, speed, and assay quality for complex biological assays
Here, we demonstrate improvement in assay sensitivity, quality, and speed of acquisition with the ImageXpress® Confocal HT.ai, our high-content imaging laser-based system.
Application Note
High-content phenotypic profiling using the Cell Painting assay
High-content phenotypic profiling using the Cell Painting assay
Here, we present a complete workflow for a cell painting assay that can be easily implemented using the ImageXpress Micro system and image analysis with machine learning capabilities
Citations
Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes
Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes
In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profil…
Blog
Life sciences technology predictions for 2021
Life sciences technology predictions for 2021
For over 30 years, Molecular Devices has been at the forefront of technological advances which have contributed to significant scientific breakthroughs. To kick off the new year, we…
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Get to know our Field Applications Scientist: Kayla Hill
Get to know our Field Applications Scientist: Kayla Hill
Kayla Hill discusses the latest trends in cellular imaging We recently hosted a webinar with our Field Applications Scientist, Kayla Hill, PhD, who explored high-content analysis…
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Imaging system produces human cellular morphological data for COVID-19 research
Imaging system produces human cellular morphological data for COVID-19 research
In an effort to better understand the cellular responses to COVID-19, the digital biology company Recursion has publicly released the world’s largest imaging dataset portraying therapeutic c…
Data Sheet
MetaXpress Software Multi-Wavelength Cell Scoring Application Module
MetaXpress Software Multi-Wavelength Cell Scoring Application Module
Multi-Wavelength Cell scoring datasheet describes the MetaXpress App Module that makes a number of additional cell-by-cell measurements including per-wavelength count.
Data Sheet
MetaXpress Software Multi-Wavelength Translocation Application Module
MetaXpress Software Multi-Wavelength Translocation Application Module
Multi-Wavelength Translocation Module is designed for the combination, or multiplexing, of multiple translocating probes into a single assay. Explore this document to know more.
Application Note
Multiplexed high-content hepatotoxicity assays using iPSC-derived hepatocytes
Multiplexed high-content hepatotoxicity assays using iPSC-derived hepatocytes
Drug-induced hepatotoxicity is an important cause for liver injury and acute liver failure. Thus highly predictive assays for safety and efficacy testing are crucial for improving drug…