A Unified Protocol to Streamline Molecular and Cellular Analysis for Three-Dimensional Cell Cultures

Three-dimensional (3D) cell cultures based on reconstituted basement membrane materials recapitulate features of extracellular matrix (ECM) and tissue stiffness in vivo and provide a physiologically relevant platform to study complex cellular processes, such as stem cell differentiation and tissue morphogenesis, that are otherwise difficult in animal models. The form and composition of 3D matrices in culture can interfere with and pose challenges for different experimental setups and assays, which necessitate alterations to facilitate analysis. Here, we provide a unified protocol for 3D cell cultures with modular workflows that streamline procedures for compatibility with common molecular and cellular assays such as live-cell imaging, immunofluorescence , qPCR, RNAseq, western blotting, and quantitative mass spectrometry.