Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects

John P. Nolan1

1 Scintillon Institute, San Diego, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.14
DOI:  10.1002/0471142956.cy1314s73
Online Posting Date:  July, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Evidence suggests that extracellular vesicles (EVs) can play roles in physiology and pathology, providing impetus to explore their use as diagnostic and therapeutic targets. However, EVs are also small, heterogeneous, and difficult to measure, and so this potential has not yet been realized. The development of improved approaches to EV detection and characterization will be critical to further understanding their roles in physiology and disease. Flow cytometry has been a popular tool for measuring cell‐derived EVs, but has often been used in an uncritical manner in which fundamental principles and limitations of the instrument are ignored. Recent efforts to standardize procedures and document the effects of different methodologies have helped to address this shortcoming, but much work remains. In this paper, I address some of the instrument, reagent, and analysis considerations relevant to measurement of individual EVs in flow, with the aim of clarifying a path to quantitative and standardized measurement of these interesting and potentially important biological nanoparticles. © 2015 by John Wiley & Sons, Inc.

Keywords: exosome; ectosome; microvesicle; microparticle; FACS

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Challenges of Extracellular Vesicle (EV) Characterization
  • Flow Cytometry of Extracellular Vesicles and Other Small Particles
  • EV Flow Cytometry: Fundamental Principles
  • Triggers and Thresholding
  • Summary and Prospects
  • Acknowledgements
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Abrams, C.S., Ellison, N., Budzynski, A.Z., and Shattil, S.J. 1990. Direct detection of activated platelets and platelet‐derived microparticles in humans. Blood 75:128‐138.
  Admyre, C., Grunewald, J., Thyberg, J., Gripenbäck, S., Tornling, G., Eklund, A., Scheynius, A., and Gabrielsson, S. 2003. Exosomes with major histocompatibility complex class II and co‐stimulatory molecules are present in human BAL fluid. Eur. Respir. J. 22:578‐583.
  Antal‐Szalmas, P., Strijp, J.A., Weersink, A.J., Verhoef, J., and Van Kessel, K.P. 1997. Quantitation of surface CD14 on human monocytes and neutrophils. J. Leukoc. Biol. 61:721‐728.
  Ardhammar, M., Lincoln, P., and Nordén, B. 2002. Invisible liposomes: Refractive index matching with sucrose enables flow dichroism assessment of peptide orientation in lipid vesicle membrane. Proc. Natl. Acad. Sci. U.S.A. 99:15313‐15317.
  Arraud, N., Gounou, C., Linares, R., and Brisson, A.R. 2014a. A Simple flow cytometry method improves the detection of phosphatidylserine‐exposing extracellular vesicles. J. Thromb. Haemost. 13:237‐247.
  Arraud, N., Linares, R., Tan, S., Gounou, C., Pasquet, J.M., Mornet, S., and Brisson, A.R. 2014b. Extracellular vesicles from blood plasma: Determination of their morphology, size, phenotype and concentration. J. Thromb. Haemost. 12:614‐627.
  Ayers, L., Kohler, M., Harrison, P., Sargent, I., Dragovic, R., Schaap, M., Nieuwland, R., Brooks, S.A., and Ferry, B. 2011. Measurement of circulating cell‐derived microparticles by flow cytometry: Sources of variability within the assay. Thromb. Res. 127:370‐377.
  Bigos, M. 2007. Separation index: An easy‐to‐use metric for evaluation of different configurations on the same flow cytometer. Curr. Protoc. Cytom. 40:1.21.1‐1.21.6.
  Bissels, U., Wild, S., Tomiuk, S., Holste, A., Hafner, M., Tuschl, T., and Bosio, A. 2009. Absolute quantification of microRNAs by using a universal reference. RNA 15:2375‐2384.
  Blessing, T., Remy, J.‐S., and Behr, J.‐P. 1998. Monomolecular collapse of plasmid DNA into stable virus‐like particles. Proc. Natl. Acad. Sci. U.S.A. 95:1427‐1431.
  Böing, A.N., van der Pol, E., Grootemaat, A.E., Coumans, F.A., Sturk, A., and Nieuwland, R. 2014. Single‐step isolation of extracellular vesicles by size‐exclusion chromatography. J. Extracell. Vesicles 3:23430. doi:10.3402/jev.v3.23430
  Chandler, W.L., Yeung, W., and Tait, J.F. 2011. A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer. J. Thromb. Haemost. 9:1216‐1224.
  Chang, C.P., Zhao, J., Wiedmer, T., and Sims, P.J. 1993. Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine. J. Biol. Chem. 268:7171‐7178.
  Chase, E.S. and Hoffman, R.A. 1998. Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity. Cytometry 33:267‐279.
  Clayton, A., Court, J., Navabi, H., Adams, M., Mason, M.D., Hobot, J.A., Newman, G.R., and Jasani, B. 2001. Analysis of antigen presenting cell derived exosomes, based on immuno‐magnetic isolation and flow cytometry. J. Immunol. Methods 247:163‐174.
  Cocucci, E., Racchetti, G., and Meldolesi, J. 2009. Shedding microvesicles: Artefacts no more. Trends Cell Biol. 19:43‐51.
  Colhoun, H.M., Otvos, J.D., Rubens, M.B., Taskinen, M.R., Underwood, S.R., and Fuller, J.H. 2002. Lipoprotein subclasses and particle sizes and their relationship with coronary artery calcification in men and women with and without type 1 diabetes. Diabetes 51:1949‐1956.
  Colombo, M., Raposo, G., and Théry, C. 2014. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30:255‐289.
  Coumans, F.A.W., van der Pol, E., Böing, A.N., Hajji, N., Sturk, G., van Leeuwen, T.G., and Nieuwland, R. 2014. Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing. J. Extracell. Vesicles 3:25922.
  Dachary‐Prigent, J., Freyssinet, J., Pasquet, J., Carron, J., and Nurden, A. 1993. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: A flow cytometry study showing a role for free sulfhydryl groups. Blood 81:2554‐2565.
  Di Vizio, D., Kim, J., Hager, M.H., Morello, M., Yang, W., Lafargue, C.J., True, L.D., Rubin, M.A., Adam, R.M., Beroukhim, R., Demichelis, F., and Freeman, M.R. 2009. Oncosome formation in prostate cancer: Association with a region of frequent chromosomal deletion in metastatic disease. Cancer Res. 69:5601‐5609.
  Di Vizio, D., Morello, M., Dudley, A.C., Schow, P.W., Adam, R.M., Morley, S., Mulholland, D., Rotinen, M., Hager, M.H., Insabato, L., Moses, M.A., Demichelis, F., Lisanti, M.P., Wu, H., Klagsbrun, M., Bhowmick, N.A., Rubin, M.A., D'Souza‐Schorey, C., and Freeman, M.R. 2012. Large oncosomes in human prostate cancer tissues and in the circulation of mice with metastatic disease. Am. J. Pathol. 181:1573‐1584.
  Dignat‐George, F. and Boulanger, C.M. 2011. The many faces of endothelial microparticles. Arterioscler. Thromb. Vasc. Biol. 31:27‐33.
  Dovichi, N.J., Martin, J.C., Jett, J.H., and Keller, R.A. 1983. Attogram detection limit for aqueous dye samples by laser‐induced fluorescence. Science 219:845‐847.
  Dovichi, N.J., Martin, J.C., Jett, J.H., Trkula, M., and Keller, R.A. 1984. Laser‐induced fluorescence of flowing samples as an approach to single‐molecule detection in liquids. Anal. Chem. 56:348‐354.
  Dragovic, R.A., Gardiner, C., Brooks, A.S., Tannetta, D.S., Ferguson, D.J., Hole, P., Carr, B., Redman, C.W., Harris, A.L., and Dobson, P.J. 2011. Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine 7:780‐788.
  Erdbrügger, U., Rudy, C.K., Etter, M.E., Dryden, K.A., Yeager, M., Klibanov, A.L., and Lannigan, J. 2014. Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry. Cytometry A 85:756‐770.
  Fay, S.P., Posner, R.G., Swann, W.N., and Sklar, L.A. 1991. Real‐time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry. Biochemistry 30:5066‐5075.
  Gardiner, C., Ferreira, Y.J., Dragovic, R.A., Redman, C.W., and Sargent, I.L. 2013. Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J. Extracell. Vesicles 2:19671.
  Gardiner, C., Shaw, M., Hole, P., Smith, J., Tannetta, D., Redman, C.W., and Sargent, I.L. 2014. Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles. J. Extracell. Vesicles 3:25361.
  Gilbert, G.E., Sims, P.J., Wiedmer, T., Furie, B., Furie, B.C., and Shattil, S.J. 1991. Platelet‐derived microparticles express high affinity receptors for factor VIII. J. Biol. Chem. 266:17261‐17268.
  Graves, S.W., Woods, T.A., Kim, H., and Nolan, J.P. 2005. Direct fluorescent staining and analysis of proteins on microspheres using CBQCA. Cytometry A 65:50‐58.
  Headland, S.E., Jones, H.R., D'Sa, A.S., Perretti, M., and Norling, L.V. 2014. Cutting‐edge analysis of extracellular microparticles using ImageStreamX imaging flow cytometry. Sci. Rep. 4:5237. doi:10.1038/srep05237
  Hercher, M., Mueller, W., and Shapiro, H.M. 1979. Detection and discrimination of individual viruses by flow cytometry. J. Histochem. Cytochem. 27:350‐352.
  Hoffman, R.A. 2009. Pulse width for particle sizing. Curr. Protoc. Cytom. 50:1.23.1‐1.23.17.
  Hoffman, R.A. and Wood, J.C.S. 2007. Characterization of flow cytometer instrument sensitivity. Curr. Protoc. Cytom. 40:1.20.1‐1.20.18.
  Hristov, M., Erl, W., Linder, S., and Weber, P.C. 2004. Apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro. Blood 104:2761‐2766.
  Jy, W., Horstman, L.L., Jimenez, J.J., Ahn, Y.S., Biro, E., Nieuwland, R., Sturk, A., Dignat‐George, F., Sabatier, F., Camoin‐Jau, L., Sampol, J., Hugel, B., Zobairi, F., Freyssinet, J.M., Nomura, S., Shet, A.S., Key, N.S., and Hebbel, R.P. 2004. Measuring circulating cell‐derived microparticles. J. Thromb. Haemost. 2:1842‐1851.
  Lacroix, R., Dubois, C., Leroyer, A.S., Sabatier, F., and Dignat‐George, F. 2013. Revisited role of microparticles in arterial and venous thrombosis. J. Thromb. Haemost. 11:24‐35.
  Lacroix, R., Robert, S., Poncelet, P., and Dignat‐George, F. 2010a. Overcoming limitations of microparticle measurement by flow cytometry. Semin. Thromb. Hemost. 36:807‐818.
  Lacroix, R., Robert, S., Poncelet, P., Kasthuri, R., Key, N., and Dignat‐George, F. 2010b. Standardization of platelet‐derived microparticle enumeration by flow cytometry with calibrated beads: Results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J. Thromb. Haemost. 8:2571‐2574.
  Lane, R.E., Korbie, D., Anderson, W., Vaidyanathan, R., and Trau, M. 2015. Analysis of exosome purification methods using a model liposome system and tunable‐resistive pulse sensing. Sci. Rep. 5:7639. doi:10.1038/srep07639
  Lee, J.A., Spidlen, J., Boyce, K., Cai, J., Crosbie, N., Dalphin, M., Furlong, J., Gasparetto, M., Goldberg, M., and Goralczyk, E.M. 2008. MIFlowCyt: The minimum information about a flow cytometry experiment. Cytometry A 73:926‐930.
  Lee, R.D., Barcel, D.A., Williams, J.C., Wang, J.G., Boles, J.C., Manly, D.A., Key, N.S., and Mackman, N. 2012. Pre‐analytical and analytical variables affecting the measurement of plasma‐derived microparticle tissue factor activity. Thromb. Res. 129:80‐85.
  Maecker, H.T. and Trotter, J. 2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037‐1042.
  Matsuzaki, K., Murase, O., Sugishita, K., Yoneyama, S., Akada, K., Ueha, M., Nakamura, A., and Kobayashi, S. 2000. Optical characterization of liposomes by right angle light scattering and turbidity measurement. Biochim. Biophys. Acta 1467:219‐226.
  Murphy, R.F. 1985. Analysis and isolation of endocytic vesicles by flow cytometry and sorting: Demonstration of three kinetically distinct compartments involved in fluid‐phase endocytosis. Proc. Natl. Acad. Sci. U.S.A. 82:8523‐8526.
  Nguyen, D.C., Keller, R.A., Jett, J.H., and Martin, J.C. 1987. Detection of single molecules of phycoerythrin in hydrodynamically focused flows by laser‐induced fluorescence. Anal. Chem. 59:2158‐2161.
  Nieuwland, R. and Sturk, A. 2010. Why do cells release vesicles? Thromb. Res. 125:S49‐S51.
  Nolan, J.P., Chambers, J.D., and Sklar, L.A. 1998. Cytometric approaches to the study of receptors. In Phagocyte Function: A Guide for Research and Clinical Evaluation (J. P. Robinson and G. F. Babcock, eds.) pp. 19‐45. Wiley‐Liss, New York.
  Nolan, J.P. and Stoner, S.A. 2013. A trigger channel threshold artifact in nanoparticle analysis. Cytometry A 83:301‐305.
  Orozco, A.F. and Lewis, D.E. 2010. Flow cytometric analysis of circulating microparticles in plasma. Cytometry A 77:502‐514.
  Ortyn, W.E., Hall, B.E., George, T.C., Frost, K., Basiji, D.A., Perry, D.J., Zimmerman, C.A., Coder, D., and Morrissey, P.J. 2006. Sensitivity measurement and compensation in spectral imaging. Cytometry A 69:852‐862.
  Rabesandratana, H., Toutant, J.‐P., Reggio, H., and Vidal, M. 1998. Decay‐accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during in vitro maturation of reticulocytes. Blood 91:2573‐2580.
  Ratajczak, M.Z. 2011. The emerging role of microvesicles in cellular therapies for organ/tissue regeneration. Nephrol. Dial. Transplant. 26:1453‐1456.
  Robert, S., Poncelet, P., Lacroix, R., Arnaud, L., Giraudo, L., Hauchard, A., Sampol, J., and Dignat‐George, F. 2009. Standardization of platelet‐derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: A first step towards multicenter studies? J. Thromb. Haemost. 7:190‐197.
  Schwartz, A., Gaigalas, A.K., Wang, L., Marti, G.E., Vogt, R.F., and Fernandez‐Repollet, E. 2004. Formalization of the MESF unit of fluorescence intensity. Cytometry B 57:1‐6.
  Shapiro, H.M. 2005. Practical Flow Cytometry. John Wiley & Sons, Hoboken, N.J.
  Sims, P.J., Faioni, E.M., Wiedmer, T., and Shattil, S.J. 1988. Complement proteins C5b‐9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity. J. Biol. Chem. 263:18205‐18212.
  Sims, P.J., Wiedmer, T., Esmon, C.T., Weiss, H.J., and Shattil, S.J. 1989. Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: An isolated defect in platelet procoagulant activity. J. Biol. Chem. 264:17049‐17057.
  Sokolova, V., Ludwig, A.‐K., Hornung, S., Rotan, O., Horn, P.A., Epple, M., and Giebel, B. 2011. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf. B Biointerfaces 87:146‐150.
  Soper, S.A., Shera, E.B., Martin, J.C., Jett, J.H., Hahn, J.H., Nutter, H.L., and Keller, R.A. 1991. Single‐molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation. Anal. Chem. 63:432‐437.
  Steen, H.B. 1980. Further developments of a microscope‐based flow cytometer: Light scatter detection and excitation intensity compensation. Cytometry 1:26‐31.
  Steen, H.B. 1986. Simultaneous separate detection of low angle and large angle light scattering in an arc lamp‐based flow cytometer. Cytometry 7:445‐449.
  Steen, H.B. 1990. Light scattering measurement in an arc lamp‐based flow cytometer. Cytometry 11:223‐230.
  Steen, H.B. 2004. Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry A 57:94‐99.
  Stoner, S.A., Duggan, E., Condello, D., Wang, S., Guerrero, A., Turk, J.R., Yan, X., Narayanan, P., and Nolan, J.P. 2015. High sensitivity flow cytometry of membrane vesicles. Cytometry A. In press.
  Théry, C., Amigorena, S., Raposo, G., and Clayton, A. 2006. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. 30:3:22.1‐3.22.29.
  Théry, C., Ostrowski, M., and Segura, E. 2009. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9:581‐593.
  Van der Heyde, H.C., Gramaglia, I., Combes, V., George, T.C., and Grau, G.E. 2011. Flow cytometric analysis of microparticles. In Flow Cytometry Protocols, 3rd Edition, Methods in Molecular Biology, Vol. 699 (T. S. Hawley and R. G. Hawley, eds.) pp. 337‐354. Humana Press, New York.
  Van der Pol, E., Hoekstra, A., Sturk, A., Otto, C., Van Leeuwen, T., and Nieuwland, R. 2010. Optical and non‐optical methods for detection and characterization of microparticles and exosomes. J. Thromb. Haemost. 8:2596‐2607.
  Van der Pol, E., van Gemert, M.J.C., Sturk, A., Nieuwland, R., and van Leeuwen, T.G. 2012. Single vs. swarm detection of microparticles and exosomes by flow cytometry. J. Thromb. Haemost. 10:919‐930.
  Van der Pol, E., Coumans, F.A.W., Grootemaat, A.E., Gardiner, C., Sargent, I.L., Harrison, P., Sturk, A., van Leeuwen, T.G., and Nieuwland, R. 2014a. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J. Thromb. Haemost. 12:1182‐1192.
  Van der Pol, E., Coumans, F.A.W., Sturk, A., Nieuwland, R., and van Leeuwen, T.G. 2014b. Refractive index determination of nanoparticles in suspension using nanoparticle tracking analysis. Nano Lett. 14:6195‐6201.
  Van der Vlist, E.J., Nolte, E.N., Stoorvogel, W., Arkesteijn, G.J., and Wauben, M.H. 2012. 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.
  Wang, L., Gaigalas, A.K., Abbasi, F., Marti, G.E., Vogt, R.F., and Schwartz, A. 2002. Quantitating fluorescence intensity from fluorophores: Practical use of MESF values. J. Res. Natl. Inst. Stand. Technol. 107:339‐354.
  Wang, L., Gaigalas, A.K., Marti, G., Abbasi, F., and Hoffman, R.A. 2008. Toward quantitative fluorescence measurements with multicolor flow cytometry. Cytometry A 73:279‐288.
  Wilson, R.B. and Murphy, R.F. 1989. Flow‐cytometric analysis of endocytic compartments. Methods Cell Biol. 31:293‐317.
  Wood, J. 1998. Fundamental flow cytometer properties governing sensitivity and resolution. Cytometry 33:260‐266.
  Wood, J. and Hoffman, R.A. 1998. Evaluating fluorescence sensitivity on flow cytometers: An overview. Cytometry 33:256‐259.
  Yuana, Y., Bertina, R.M., and Osanto, S. 2011. Pre‐analytical and analytical issues in the analysis of blood microparticles. Thromb. Haemost. 105:396‐408.
  Zarrin, F. and Dovichi, N.J. 1985a. Particle counting by laser light scatter for capillary hydrodynamic chromatography. Anal. Chem. 57:1826‐1829.
  Zarrin, F. and Dovichi, N.J. 1985b. Sub‐picoliter detection with the sheath flow cuvette. Anal. Chem. 57:2690‐2692.
  Zarrin, F. and Dovichi, N.J. 1987. Effect of sample stream radius upon light scatter distributions generated with a Gaussian beam light source in the sheath flow cuvette. Anal. Chem. 59:846‐850.
  Zarrin, F., Risfelt, J.A., and Dovichi, N.J. 1987. Light scatter detection within the sheath flow cuvette for size determination of multicomponent submicrometer particle suspensions. Anal. Chem. 59:850‐854.
  Zhu, S., Ma, L., Wang, S., Chen, C., Zhang, W., Yang, L., Hang, W., Nolan, J.P., Wu, L., and Yan, X. 2014. Light‐scattering detection below the level of single fluorescent molecules for high‐resolution characterization of functional nanoparticles. ACS Nano 8:10998‐11006.
  Zwicker, J.I. 2010. Impedance‐based flow cytometry for the measurement of microparticles. Semin. Thromb. Hemost. 36:819‐823.
  Zwicker, J.I., Trenor, C.C. III., Furie, B.C., and Furie, B. 2011. Tissue factor‐bearing microparticles and thrombus formation. Arterioscler. Thromb. Vasc. Biol. 31:728‐733.
Internet Resources
  http://www.philiplaven.com/mieplot.htm
  Philip Laven, 2013. MiePlot: A computer program for scattering of light from a sphere using Mie theory and the Debye series.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library