Analysis of Individual Extracellular Vesicles by Flow Cytometry
Extracellular vesicles (EVs) are released by cells and can be found in cell culture supernatants and biofluids. EVs carry proteins, nucleic acids, and other cellular components and can deliver these to nearby or distant cells, making EVs of interest as both disease biomarkers and therapeutic targets. EVs in biofluids are heterogeneous, coming from different cell types and from different sources with the cell, which limits the usefulness of bulk EV analysis methods that report the average features of all EVs present. Single-particle measurements such as flow cytometry would be preferred, but the small size and low abundance of surface antigens challenges conventional flow cytometry approaches, leading to the development of vesicle-specific assays and experimental design. Among the key issues that have emerged are: (a) judicious choice of detection (triggering) approach; (b) appropriate control experiments to confirm the vesicular nature of the detected events and the contribution of coincidence (aka swarm detection); and (c) the importance of fluorescence calibration to allow data to be compared over time and between laboratories. We illustrate these issues in the context of fluorescence-triggered Vesicle Flow Cytometry (VFC), a general approach to the quantitative measurement of EV number, size, and surface marker expression.
Key wordsExosome Microvesicle Calibration Standardization
Supported by: UH2TR000931 from the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director.
- 1.Colombo M, Raposo G, Théry C (2014) Biogenesis, secretion, and intercellular interactions of Exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30(1)Google Scholar
- 4.van der Pol E, Coumans FAW, Grootemaat AE, Gardiner C, Sargent IL, Harrison P, Sturk A, van Leeuwen TG, Nieuwland R (2014) 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(7):1182–1192. doi: 10.1111/jth.12602 CrossRefGoogle Scholar
- 5.Lacroix R, Judicone C, Mooberry M, Boucekine M, Key NS, Dignat-George F, the ISSCW (2013) Standardization of pre-analytical variables in plasma microparticle determination: results of the international society on thrombosis and Haemostasis SSC collaborative workshop. J Thromb Haemost 11(6):1190–1193. doi: 10.1111/jth.12207 CrossRefGoogle Scholar
- 7.Lacroix R, Robert S, Poncelet P, Kasthuri R, Key N, Dignat-George F (2010) 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(11):2571–2574CrossRefGoogle Scholar
- 8.Lötvall J, Hill AF, Hochberg F, Buzás EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Théry C (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3(1.) 3:10.3402/jev.v3403.26913). doi: 10.3402/jev.v3.26913
- 9.Witwer KW, Buzás EI, Bemis LT, Bora A, Lässer C, Lötvall J, Nolte-‘t Hoen EN, Piper MG, Sivaraman S, Skog J, Théry C, Wauben MH, Hochberg F (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles, 2. 10.3402/jev.v3402i3400.20360. doi: 10.3402/jev.v2i0.20360
- 11.Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protocols Cell Biol Unit 3.22. 21-23.22. 29Google Scholar
- 15.Robert S, Poncelet P, Lacroix R, Arnaud L, Giraudo L, Hauchard A, Sampol J, 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(1):190–197CrossRefGoogle Scholar
- 16.Cointe S, Judicone C, Robert S, Mooberry M, Poncelet P, Wauben M, Nieuwland R, Key N, Dignat-George F, Lacroix R (2016) Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop. J Thromb Haemost 15(1):187–193CrossRefGoogle Scholar
- 22.Brooks MB, Turk JR, Guerrero A, Narayanan PK, Nolan JP, Besteman EG, Wilson DW, Thomas RA, Fishman CE, Thompson KL, Eliinger-Ziegelbauer H, Pierson JB, Paulman A, Chiang AY, Schultze AE (2016) Non-lethal endotoxin injection: a rat model for new biomarkers of hypercoagulability. PLoS One 12(1):e0169976CrossRefGoogle Scholar
- 27.Nolan JP, Chambers JD, Sklar LA (1998) Cytometric approaches to the study of receptors. Phagocyte function: a guide for research and clinical evaluation. Wiley-Liss, New York, pp 19–45Google Scholar
- 28.Woods TA, Graves SW, Nolan JP (2005) Microsphere Surface Protein Determination Using Flow Cytometry. Current Protocols in Cytometry: Unit13.12. 11-13.12. 13Google Scholar