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Cell Painting PLUS: An Iterative Staining-Elution Protocol for High-Content Phenotypic Screenings.

Marlene Wedler1,2, Elena von Coburg1, Jose M Muino1

  • 1German Centre for the Protection of Laboratory Animals (Bf3R) and Experimental Toxicology, German Federal Institute for Risk Assessment (BfR), Berlin, Germany.

Current Protocols
|April 27, 2026
PubMed
Summary
This summary is machine-generated.

Cell Painting PLUS (CPP) is a novel method that uses iterative staining with seven fluorescent dyes to analyze nine cellular compartments, enhancing morphological profiling for drug screening. This method offers increased multiplexing and organelle specificity compared to existing Cell Painting techniques.

Keywords:
Cell Painting PLUS (CPP)cell‐based assayshigh‐throughput high‐content screeningsmorphological profilingnew approach methodologies (NAMs)

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Area of Science:

  • Cellular and Molecular Biology
  • High-Content Screening
  • Bioimaging

Background:

  • Cell Painting (CP) enables morphological profiling by labeling cellular compartments with fluorescent dyes.
  • Existing CP methods offer limited multiplexing and organelle specificity.
  • Comprehensive analysis of cellular phenotypes requires advanced imaging techniques.

Purpose of the Study:

  • To present a detailed protocol for Cell Painting PLUS (CPP), an advanced multiplexed imaging method.
  • To introduce an automated image and data analysis strategy for CPP.
  • To expand the multiplexing capacity and organelle specificity of CP-based screening.

Main Methods:

  • Iterative staining, elution, and re-staining with seven fluorescent dyes to label nine cellular compartments.
  • Extraction of 894 morphological features from single cells for phenotypic profiling.
  • Automated data analysis workflow including quality control, normalization, and visualization using CPPAnalyzer and CPPManager.

Main Results:

  • CPP enables multiplexed analysis of nine cellular compartments and organelles with high specificity.
  • Generated comprehensive phenotypic profiles based on 894 morphological features.
  • Developed user-friendly automated workflows (Jupyter notebook and KNIME) for data processing.

Conclusions:

  • CPP significantly enhances the multiplexing and organelle specificity of Cell Painting methods.
  • The automated analysis workflow facilitates efficient and accessible morphological profiling.
  • CPP provides a powerful tool for drug discovery and understanding cellular responses.