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Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry

Abstract

We provide a protocol for a high-resolution flow cytometry–based method for quantitative and qualitative analysis of individual nano-sized vesicles released by cells, as developed and previously described by our group. The method involves (i) bright fluorescent labeling of cell-derived vesicles and (ii) flow cytometric analysis of these vesicles using an optimized configuration of the commercially available BD Influx flow cytometer. The method allows the detection and analysis of fluorescent cell-derived vesicles of 100 nm. Integrated information can be obtained regarding the light scattering, quantity, buoyant density and surface proteins of these nano-sized vesicles. This method can be applied in nanobiology to study basic aspects of cell-derived vesicles. Potential clinical applications include the detailed analysis of vesicle-based biomarkers in body fluids and quality control analysis of (biological) vesicles used as therapeutic agents. Isolation, fluorescent labeling and purification of vesicles can be done within 24 h. Flow cytometer setup, calibration and subsequent data acquisition can be done within 2–4 h by an experienced flow cytometer operator.

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Figure 1: Flow cytometric detection of nano-sized beads and cell-derived vesicles by fluorescence-based thresholding.
Figure 2: Antibody-mediated detection of specific proteins on cell-derived vesicles.
Figure 3: Quantification of nano-sized beads and cell-derived vesicles by flow cytometric analysis.
Figure 4: Hardware adaptations of the BD Influx flow cytometer for analysis of individual nano-sized vesicles.
Figure 5: Modified BD Influx configuration for reduced wide-angle FSC detection.
Figure 6: Influx settings and sucrose gradient fraction collection setup.
Figure 7: Flow diagram of procedure steps.

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Acknowledgements

We thank H.C.H. Mertens, B.J. Bosch, W. Bartelink, E. Mastrobattista, E.V.B. van Gaal, M. Aalberts (all from Utrecht University, Utrecht) and R.E. Mebius (Vrije Universiteit Medisch Centrum, Amsterdam) for providing reagents, experimental support and/or valuable discussion for validation of the method, and H. de Boer-Brouwer for technical assistance. We thank T. van Haeften, R.W. Wubbolts and J. van der Lit for critical reading of the manuscript.

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E.J.v.d.V. and E.N.M.N.-'t H. designed and performed all experiments, analyzed data and wrote the paper. W.S. gave technical and conceptual advice and edited the manuscript. G.J.A.A. optimized the configuration and performance of the flow cytometer, performed experiments and wrote the paper. M.H.M.W. initiated and supervised the research, designed experiments and wrote the paper. G.J.A.A. and M.H.M.W. contributed equally as senior author.

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Correspondence to Marca H M Wauben.

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van der Vlist, E., Nolte-'t Hoen, E., Stoorvogel, W. et al. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protoc 7, 1311–1326 (2012). https://doi.org/10.1038/nprot.2012.065

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