Abstract
Well-characterized small molecules enable the study of cell processes and facilitate target validation. Here, we describe a high-content multiplex screen to investigate cell viability over 48 h, which can be combined with investigating phenotypic features, such as tubulin binding and mitochondrial content, as initial cellular quality control of diverse compounds. The protocol is on a live-cell basis and easily adaptable and scalable. It details cell preparation, compound handling, plate layout configuration, image acquisition with the CQ1, and data analysis using the CellPathfinder software. For complete details on the use and execution of this protocol, please refer to Tjaden et al. (2022).