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Short answer: Cell Painting is an image-based morphological profiling assay. Cells in multiwell plates are stained with six fluorescent dyes, imaged in five channels, and measured for roughly 1,500 morphological features per cell, so that the effect of a chemical or genetic perturbation can be compared with the effect of others (Bray et al., 2016).
Definition and scope
The assay was described as a protocol in Nature Protocols in 2016. Its six dyes reveal eight cellular components or organelles, and automated image analysis extracts a numeric profile for each cell. The profile, not any single stained structure, is the readout. A later 2023 protocol update (Cimini et al.) describes the method as introduced in 2013 and notes its use for clustering perturbations, describing disease phenotypes and machine-learning prediction.
It is a lab-technique topic for researchers and core-facility staff. It belongs with other protocol and imaging topics such as epifluorescence microscopy and cell culture basics, and the plate-level record keeping behind it is the kind of thing a standard operating procedure is meant to fix.
How it is used in practice
The 2016 protocol is built around 384-well plates. Its stated timeline is about two weeks for cell culture and imaging, followed by one to two further weeks for feature extraction and data analysis. The 2023 update gives the same one-to-two-week ranges for culture and imaging of up to 20 plates, then one to two weeks of analysis.
The protocol names two main applications:
- Mechanism of action of compounds: a compound’s profile is matched against profiles of annotated compounds.
- Gene function: genetic perturbations that give similar profiles are grouped as potentially related in function.
What each dye stains
The table is taken from Table 1 of the 2016 protocol and cross-checked against the component list in the 2023 update.
| Dye or conjugate | Labels |
|---|---|
| Hoechst 33342 | Nucleus (DNA) |
| Concanavalin A / Alexa Fluor 488 conjugate | Endoplasmic reticulum |
| SYTO 14 | Nucleoli and cytoplasmic RNA |
| Phalloidin / Alexa Fluor 568 conjugate | F-actin cytoskeleton |
| Wheat germ agglutinin (WGA) / Alexa Fluor 555 conjugate | Golgi and plasma membrane |
| MitoTracker Deep Red | Mitochondria |
Version 1 versus version 3
The 2023 update reports a quantitative optimisation by the JUMP Cell Painting Consortium. Its findings, as stated in the abstract: dyes from two different vendors work equivalently well, version 3 simplifies some steps, and several stain concentrations can be reduced compared with the original protocol.
Where it matters for labs
The JUMP Cell Painting Consortium describes itself as an image-based drug discovery effort, and states that its data are now public through the JUMP Hub. Anyone planning an assay should treat the following limitations, listed in the 2016 protocol, as a checklist:
- RNAi reagents can have off-target effects.
- Some biological processes show no phenotype under the chosen stains, cell type or time point.
- Overfitting and multiple testing are risks when many features are compared.
- Single-cell data are large, so storage and compute needs are significant.
- Batch effects can arise between experiments.
Good reproducibility practice and a data management plan cover the storage and batch-tracking points.
Common misunderstandings
- One dye per organelle. Not so: six dyes label eight components. SYTO 14 marks both nucleoli and cytoplasmic RNA, and WGA marks both the Golgi and the plasma membrane.
- It identifies a mechanism by itself. The protocol describes inference by matching to annotated profiles, not direct measurement of mechanism.
- It detects every effect. The authors state that some processes produce no phenotype under the chosen conditions.
FAQ
What is Cell Painting?
A multiplexed fluorescence imaging assay that stains eight cellular components with six dyes and extracts about 1,500 features per cell for morphological profiling.
How long does a Cell Painting experiment take?
The published protocols give about one to two weeks for cell culture and imaging, plus one to two weeks for feature extraction and analysis.
Is the data public?
The JUMP Cell Painting Consortium states that its data are public via the JUMP Hub.
Sources
- Bray M-A et al. Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes. Nature Protocols 2016;11:1757-1774. PMC5223290 (read 2026-11-08).
- Cimini BA et al. Optimizing the Cell Painting assay for image-based profiling. Nature Protocols 2023;18:1981-2013. PMID 37344608 (abstract read 2026-11-08).
- JUMP Cell Painting Consortium. jump-cellpainting.broadinstitute.org (read 2026-11-08).
