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Labeling, isolation and characterization of cell-type-specific exosomes derived from mouse skin tissue

Nature Protocols volume 21, pages 1192–1234 (2026)Cite this article

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Abstract

Extracellular vesicles are a heterogeneous group of membrane-bound vesicles involved in cell–cell communication, formed at the plasma membrane (ectosomes) or by endocytosis (exosomes). Most exosome studies so far have focused on in vitro systems or exosomes derived from bodily fluids, while tissue-derived exosomes remain underexplored. Here we present a protocol using cell-type-specific promoter-driven reporter constructs for the targeted labeling and subsequent isolation of exosomes from specific cell types in vivo from mouse tissues. The differentiation between exosomes and ectosomes remains challenging due to limitations of current isolation techniques that are primarily based on size, density or surface markers. To address this issue, our approach leverages genetic engineering to mark exosomes specifically, enabling their precise identification and isolation from a complex biological pool of heterogenous extracellular vesicles. The isolated cell-type-specific exosomes are characterized by electron microscopy, nanoparticle tracking analysis, antibody exosome array assay and other established techniques. The labeling and isolation of exosomes spans 2–3 days and is designed to be accessible to researchers with fundamental laboratory competencies. This protocol facilitates the study of exosome-mediated cellular communication by enabling the isolation of cell-type-specific exosomes from either individual cell types or multiple cell types in combination. Most experiments within the protocol have used murine wound-edge skin tissue, but the protocol can, in principle, also be applied to other tissues to isolate exosomes, with a few modifications as required. This methodology opens new avenues for exploring the functional roles of cell-type-specific exosomes in intercellular communication.

Key points

  • Exosomes—extracellular vesicles of endosomal origin—are important mediators of cell–cell communications yet are difficult to isolate from the heterogeneous pool of extracellular vesicles using methods based on size, density and surface markers.

  • This protocol uses tissue nanotransfection to deliver reporter constructs driven by cell-type-specific promoters for the immunomagnetic isolation of specific exosome populations from mouse tissue followed by elution with acidic buffer and purification by ultracentrifugation. The resulting exosomes are characterized by established assays.

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Fig. 1: A plasmid construct for labeling and isolation of Exoκ-GFP.
Fig. 2: A schematic representation of the steps involved in isolating Exoκ-GFP from tissue.
Fig. 3: A visualization of exosomes originating from different cell types in mouse skin tissue.
Fig. 4: The characterization of Exoκ-GFP.
Fig. 5: The transformation of the plasmid construct for labeling and isolation of Exoκ-GFP in competent E. coli DH5α cells.
Fig. 6: The promoter specificity of the plasmid construct for labeling and isolation of Exoκ-GFP.
Fig. 7: A schematic representation of the steps involved in testing promoter specificity of plasmid construct for labeling and isolation of Exoκ-GFP.
Fig. 8: The in vivo delivery of plasmid construct for the labeling and isolation of Exoκ-GFP via TNT.
Fig. 9: The visualization and abundance of Exoκ-GFP in mouse wound-edge tissue.

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Data availability

The authors declare that the main data discussed in this protocol are available in the supporting primary research paper53. The snapgene files, QC files and service reports of the plasmids are available at Figshare via https://doi.org/10.6084/m9.figshare.26169487.v1 (ref. 99).

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Acknowledgements

The TNT chips were fabricated at the Pritzker Nanofabrication Facility, which receives partial support from the SHyNE Resource, a node of the National Science Foundation’s National Nanotechnology Coordinated Infrastructure (grant no. NSF ECCS-2025633). This work was supported by NIH (grant no. DK129592 to S.G. and grant no. GM143572 to Y.X. and in part by grant nos. DK128845 and DK135447 to C.K.S.).

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Author notes

  1. These authors contributed equally: Anita Yadav, Anu Sharma.

Authors and Affiliations

  1. McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA

    Anita Yadav, Anu Sharma, Mohini Moulick, Parmeshwar V. Gavande, Aparajita Nandy, Yi Xuan, Chandan K. Sen & Subhadip Ghatak

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Contributions

A.Y. and A.S. designed and performed the TNT procedure on mice and other biological experiments. M.M., P.V.G. and A.N. carried out characterization. Y.X. fabricated and performed quality control on the TNT chip. C.K.S. provided resources and supervision. S.G. conceived of the idea, provided plasmid design guidelines to ABMGood, supervised and led this project. All authors have contributed to writing and editing the manuscript.

Corresponding author

Correspondence to Subhadip Ghatak.

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The authors declare no competing interests.

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Nature Protocols thanks Rossella Crescitelli, Ke Cheng and Saman Yasamineh for their contribution to the peer review of this work.

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Key references

Zhou, X. et al. ACS Nano 14, 12732–12748 (2020): https://doi.org/10.1021/acsnano.0c03064

Brown, B. A. et al. Anal. Chem. 94, 8909–8918 (2022): https://doi.org/10.1021/acs.analchem.2c00453

Sharma, A. et al. ACS Nano 18, 30405–30420 (2024): https://doi.org/10.1021/acsnano.4c07610

Xuan, Y. et al. Nat. Protoc. 16, 5707–5738 (2021): https://doi.org/10.1038/s41596-021-00631-0

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Yadav, A., Sharma, A., Moulick, M. et al. Labeling, isolation and characterization of cell-type-specific exosomes derived from mouse skin tissue. Nat Protoc 21, 1192–1234 (2026). https://doi.org/10.1038/s41596-025-01238-5

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