The Cell Painting PLUS Method: Phenotypic Profiling Using Iterative Staining-Elution Cycles
Elena von Coburg1,2, Sebastian Dunst3
1German Centre for the Protection of Laboratory Animals (Bf3R) and Experimental Toxicology, German Federal Institute for Risk Assessment (BfR), Berlin, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|January 1, 2026
Summary
Cell Painting PLUS (CPP) enhances high-throughput phenotypic profiling by enabling iterative cell staining and imaging. This robust method offers greater flexibility for detailed cellular analysis in drug discovery and toxicology.
Area of Science:
- Cellular imaging
- High-throughput screening
- Phenotypic profiling
Background:
- Cell Painting (CP) is a standard imaging technique for high-throughput phenotypic profiling (HTPP).
- Standard CP has limitations in flexibility and multiplexing capabilities for detailed cellular analysis.
- There is a need for advanced HTPP methods to overcome existing boundaries.
Purpose of the Study:
- Introduce Cell Painting PLUS (CPP), an advanced HTPP method.
- Demonstrate CPP's efficiency, robustness, and broad applicability.
- Highlight CPP's enhanced flexibility in dye selection and multiplexing for phenotypic profiling.
Main Methods:
- Utilizes an elution buffer for iterative staining, elution, and re-staining of the same cells.
- Employs at least seven fluorescent dyes to label nine cellular compartments.
- Automated staining and imaging procedures coupled with Harmony software for image analysis.
Main Results:
- CPP offers higher flexibility in selecting and combining fluorescent dyes for customized multiplexing.
- Separate imaging and analysis of single dyes ensure high specificity of phenotypic profiles.
- Enables precise insights into cellular processes and dysfunction.
Conclusions:
- CPP overcomes limitations of standard CP, providing a more flexible HTPP approach.
- The method allows for unbiased and in-depth characterization of compound or genetic perturbation effects.
- CPP is valuable for toxicology and biomedical research, aiding in understanding modes-of-action.
Keywords:
Cell Painting PLUSHarmony softwareHigh-content screeningHigh-throughputMorphological fingerprintsPhenotypic profilingMore Related Videos
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