This reference guide outlines the Key Considerations for Live Cell Painting (LCP) screens applied to High-Throughput Screening (HTS). LCP offers superior biological relevance by capturing cells in their most physiological, unperturbed state, compared to traditional fixed-cell methods.

To successfully adopt LCP for HTS, a new workflow is required that ensures non-perturbing and scalable data acquisition. Essential considerations include standardized cell management (bank & passage), compound addition, imaging optimization, and rigorous assay validation.

The results show that LCP HTS is feasible, achieving throughput comparable to fixed-cell methods when utilizing optimized steps, including non-toxic imaging. The key advantage of LCP is the superior biological relevance, additional kinetic data and a simpler, less hands-on workflow (mix-and-read, no fixation). This method provides drug discovery groups with a scalable approach for phenotypic profiling in live cells.