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Fluorescence-activated particle sorting for condensate purification

Nature Protocols (2025)Cite this article

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Abstract

Condensates are receiving increasing attention because of their ability to organize subcellular space. In eukaryotes, nuclear condensates include nucleoli and paraspeckles, and cytoplasmic ones include P-bodies (PBs) and stress granules. One approach to investigate condensate biology is through analyzing their protein and RNA content. However, purifying condensates remains a challenge because of their densities being similar to various other organelles, and the absence of protein markers accumulating exclusively in them. These limitations, combined with the generally low number of condensates per cell, necessitate new approaches to tackle their purification. Here, we present a protocol describing fluorescence-activated particle sorting (FAPS) for purifying condensates. In brief, FAPS involves fluorescently labeling condensates to identify and isolate them from other cellular components via sorting. In this Protocol, we focus on PB purification, quality control and downstream characterization of PB protein and RNA contents. Although originally developed to purify PBs from human cell lines, FAPS can be adapted to various condensates across model organisms. The procedure requires knowledge in basic cell culture, molecular biology and flow cytometry and access to a fluorescence-activated cell sorter with sufficient sensitivity. It requires ~25–30 d, including a hands-on period of 15 d, to complete. In summary, FAPS allows the characterization of the content of diverse condensates across cell types and organisms.

Key points

  • FAPS is a sorting-based method to purify fluorescently labeled condensates from a cellular lysate by taking advantage of simple sorting controls. This allows the investigation of condensate biology through characterization of their nucleic acid and protein contents.

  • FAPS overcomes limitations of approaches used in yeast, which rely on a combination of cross-linking, fractionation and affinity purification of condensate-enriched proteins, allowing the purification of condensates from mammalian cells.

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Fig. 1: Overview of FAPS.
Fig. 2: Cell lines and lysates used in FAPS.
Fig. 3: Particle sorting example.
Fig. 4: Example of sample organization across a FAPS pipeline.
Fig. 5: Mass spectrometry analysis of the protein content of PBs.
Fig. 6: Analysis of the RNA content of PBs.

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Data availability

The raw data presented in this Protocol are available in the supporting primary research papers. Raw RNA-seq data of PBs purified across the cell cycle (used in Fig. 6) are available at ArrayExpress under the accession number E-MTAB-12923. Further details are available from the corresponding author upon request.

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Acknowledgements

A.S. was supported by the Agence Nationale pour la Recherche (ANR) grant no. ANR-19-CE12-0024-01 and INCa grant 2022-082. The work was supported by the grants ANR-19-CE12-0024 and INCa 2022-082 (to D.W.). We thank M. Bénard, M.-N. Benassy and M. Ernoult-Lange for critical reading of the manuscript and A. Cornu for discussions on particle sorting.

Author information

Authors and Affiliations

  1. Research Center Saint-Antoine (CRSA), CISA Flow Cytometry Facility, UMRS 938, Sorbonne University, Paris, France

    Annie Munier Godebert

  2. Sorbonne Université, CNRS, INSERM, Development, Adaptation and Ageing, Dev2A, Paris, France

    Dominique Weil & Adham Safieddine

  3. Sorbonne Université, CNRS, INSERM, Institut de Biologie Paris-Seine, IBPS, Paris, France

    Dominique Weil & Adham Safieddine

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  1. Annie Munier Godebert
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  2. Dominique Weil
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Contributions

A.S. and D.W. conceptualized the study. A.S. and A.M.G. set up the sorting. D.W. acquired funding. A.S. wrote the original draft. All authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Adham Safieddine.

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Key references

Safieddine, A. et al. Mol. Cell 84, 4191–4208.e7 (2024): https://doi.org/10.1016/j.molcel.2024.09.011

Hubstenberger, A. et al. Mol. Cell 68, 144–157.e5 (2017): https://doi.org/10.1016/j.molcel.2017.09.003

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Munier Godebert, A., Weil, D. & Safieddine, A. Fluorescence-activated particle sorting for condensate purification. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01216-x

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