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

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.