This document outlines the general workflow for Live Cell Painting use cases, focusing on the methodology for analysis of ChromaLIVE™ data:

• Image Analysis: Cells are segmented and features extracted.

• Data processing: Features are transformed and normalized to mitigate batch effects.

• Data aggregation & dimensionality reduction: Single-cell data is aggregated and reduced for visualization.

• Phenotypic profiling: Profile similarity metrics are used to cluster compounds by MoA.

• Key Endpoints: This workflow supports assay Validation and dose-response determination. It leverages various analysis softwares for robust high-content imaging data analysis.