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Studying extracellular vesicle transfer by a Cre-loxP method

Nature Protocols volume 11, pages 87–101 (2016)Cite this article

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Abstract

Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro– or ex vivo–isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase–expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre+ cells and reporter+ cells, which takes ∼6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to ∼2 months.

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Figure 1: The Cre-loxP method for studying EV transfer in vitro and in vivo.
Figure 2: The generation of Cre+ and reporter+ cell lines.
Figure 3: Visualization and quantification of in vitro EV transfer.
Figure 4: Studying the functions of in vivo EV transfer.

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Acknowledgements

We thank A. de Graaff and the Hubrecht Imaging Centre for imaging support. We thank A. Kamermans for experimental and imaging assistance and W. Karthaus for his help with cloning the pLV-CMV-LoxP-DsRed-LoxP-eGFP construct. We thank J. Segall (Albert Einstein College of Medicine, New York) for providing us with MDA-MB-231 cells. This work was supported by a Vidi fellowship (no. 91710330); research grants from the Dutch Organization of Scientific Research (NWO; no. 823.02.017), the Dutch Cancer Society (KWF; HUBR 2009-4621) and Cancer Genomics Netherlands (all to J.v.R.); and equipment grants (nos. 175.010.2007.00 and 834.11.002) from the Dutch Organization of Scientific Research (NWO).

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Author notes

  1. Anoek Zomer and Sander Christiaan Steenbeek: These authors contributed equally to this work.

Authors and Affiliations

  1. Cancer Genomics Netherlands, Hubrecht Institute-Royal Netherlands Academy of Sciences (KNAW) and University Medical Centre Utrecht, Utrecht, the Netherlands

    Anoek Zomer, Sander Christiaan Steenbeek, Carrie Maynard & Jacco van Rheenen

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  1. Anoek Zomer
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  2. Sander Christiaan Steenbeek
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  3. Carrie Maynard
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Contributions

A.Z. and S.C.S. wrote the manuscript, prepared the figures and optimized the described procedures and the data presented. C.M. optimized the Transwell experiments and helped to write the manuscript. J.v.R. conceived and supervised the study, and helped to write the manuscript.

Corresponding author

Correspondence to Jacco van Rheenen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Visualizing uptake of in vitro-purified Cre+ EVs

EVs isolated from Cre+ C26 colorectal carcinoma cells were isolated using differential centrifugation as described in the Supplementary Methods. Different amounts of these EVs were added to C26 reporter+ cells and after 72 h incubation, eGFP+ cells were scored by fluorescence microscopy (n = 1).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and Supplementary Methods (PDF 304 kb)

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Zomer, A., Steenbeek, S., Maynard, C. et al. Studying extracellular vesicle transfer by a Cre-loxP method. Nat Protoc 11, 87–101 (2016). https://doi.org/10.1038/nprot.2015.138

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