Abstract
Biomolecular condensates are essential for cellular organization, yet their formation dynamics and molecular content exchange properties remain poorly understood. Here we show that flow cytometry provides a high-throughput, solution-based platform for analyzing condensate behavior at the single-droplet level. Using self-interacting NPM1 condensates as a model, we demonstrate that this approach quantifies phase behavior across protein and salt conditions, measures the partitioning of diverse macromolecules—including antibodies, lipids, small-molecule drugs, and RNA—and detects molecular colocalization with high statistical precision. Importantly, we establish a high-throughput assay to track real-time molecular exchange between preformed condensates and newly added, orthogonally tagged protein. These measurements reveal that condensate aging significantly reduces molecular dynamisms, likely due to altered biophysical properties with time. Compared to conventional imaging techniques that require surface immobilization or complex instrumentation, our method enables rapid, quantitative characterization of condensate dynamics and molecular content, providing a scalable framework for probing condensate function.
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Data availability
All flow cytometry data (FCS files) are available at Zenodo under: https://zenodo.org/records/17279422. Source data for all main-text figures and Supplementary Figs. are provided in the accompanying Source Data file. The full coarse-grained (CG) molecular dynamics trajectory files exceed public repository size limits and are therefore available under restricted access for file-size reasons; access can be obtained by contacting the corresponding author (M.M.P.) Source data are provided with this paper.
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Acknowledgements
This study was supported by the NIH grants 1R01AI178975-01 (M.M.P.), R35GM124893 (M.M.P.), R35GM153388 (J.M.), R01AI179080-01 (M.M.P.). We thank the W.M. Keck Center for Cellular Imaging for the usage of Zeiss LSM 980 microscopy System and Leica STELLARIS 8 confocal/FLIM/tauSTED microscope system (NIH OD030409) and thank the Flow Cytometry Core Facility for the usage of Amnis ImageStreamX Mark II system. We acknowledge the Texas A&M High Performance Research Computing (HPRC) for providing computational resources that have contributed to the results reported in the article. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
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Y.H., G.M.O., and M.M.P. conceived and designed the study. Y.H. and G.M.O. performed all flow cytometry assays, imaging flow cytometry (IFC), and data analysis. Y.H. conducted confocal imaging and turbidity measurements. Y.H. and J.Mo. carried out protein expression and purification. A.M. performed the coarse-grained (CG) molecular dynamics simulations and theoretical analyses under the supervision of J.Mi. M.M.P. provided project oversight and conceptual guidance. Y.H. and M.M.P. wrote the manuscript with input from all authors. All authors reviewed and approved the final version of the manuscript.
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He, Y., Ongwae, G.M., Mondal, A. et al. A high-throughput, flow cytometry approach to measure phase behavior and exchange in biomolecular condensates. Nat Commun (2026). https://doi.org/10.1038/s41467-025-68093-6
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DOI: https://doi.org/10.1038/s41467-025-68093-6
