A High-Throughput, Flow Cytometry Approach to Measure Phase Behavior and Exchange in Biomolecular Condensates

Biomolecular condensates are essential for cellular organization, yet their formation dynamics and molecular content exchange properties remain poorly understood. In this study, we developed a high-throughput flow cytometry approach to quantify condensate formation, molecular colocalization, and dynamic exchange. Using self-interacting NPM1 condensates as a model system, we benchmarked the use of flow cytometry to broadly characterize their phase behavior across various protein concentrations and salt conditions. We further demonstrated that flow cytometry can assess the colocalization of macromolecules - including antibodies, lipids, small-molecule drugs, and RNAs, within NPM1 condensates. Importantly, we established the first assay to track real-time molecular exchange between preformed condensates and newly added, orthogonally tagged protein. This revealed that condensate aging significantly reduces molecular dynamisms, likely due to altered biophysical properties with time. Compared to conventional imaging techniques, which often require immobilized samples and complex experimental setups, our solution-based approach enables rapid, quantitative analysis of condensate behavior at the single-droplet level. This work provides a powerful framework for studying biomolecular condensates with enhanced precision and scalability, offering new insights into their dynamic properties and molecular interactions.