Abstract
Fluorescence nanoparticle tracking analysis (fl-NTA) allows for accurate sizing, counting, and phenotyping of extracellular vesicles (EV). Here, we present two protocols for the analysis of EVs using fl-NTA, highlighting the potential pitfalls and challenges. The first protocol utilizes CellMask Orange™ (CMO) as a general membrane marker to label EVs derived from plasma. The second protocol describes the use of a Qdot-conjugated antibody to identify syncytiotrophoblast (STB)-derived EVs. “Standard” preparations of STB-derived EVs enriched for either microvesicles (STBMV) or exosomes (STBEX), containing a known amount of EV positive for the STB specific antigen placental alkaline phosphatase (PLAP), were also used to optimize fl-NTA camera settings.
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van der Pol E, Coumans F, Varga Z et al (2013) Innovation in detection of microparticles and exosomes. J Thromb Haemost 11(Suppl 1):36–45. doi:10.1111/jth.12254
van der Pol E, Hoekstra AG, Sturk A et al (2010) Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost 8(12):2596–2607. doi:10.1111/j.1538-7836.2010.04074.x
Witwer KW, Buzas EI, Bemis LT et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2. doi:10.3402/jev.v2i0.20360
Dragovic RA, Gardiner C, Brooks AS et al (2011) Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine 7(6):780–788. doi:10.1016/j.nano.2011.04.003
Dragovic RA, Collett GP, Hole P et al (2015) Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis. Methods 87:64–74. doi:10.1016/j.ymeth.2015.03.028. http://creativecommons.org/licenses/by/4.0/
Gardiner C, Ferreira YJ, Dragovic RA et al (2013) Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles 2. doi:10.3402/jev.v2i0.19671
Tannetta DS, Dragovic RA, Gardiner C et al (2013) Characterisation of syncytiotrophoblast vesicles in normal pregnancy and pre-eclampsia: expression of Flt-1 and endoglin. PLoS One 8(2):e56754. doi:10.1371/journal.pone.0056754
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This work was supported by MRC programme grant MR/J003360/1.
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Carnell-Morris, P., Tannetta, D., Siupa, A., Hole, P., Dragovic, R. (2017). Analysis of Extracellular Vesicles Using Fluorescence Nanoparticle Tracking Analysis. In: Kuo, W., Jia, S. (eds) Extracellular Vesicles. Methods in Molecular Biology, vol 1660. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7253-1_13
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DOI: https://doi.org/10.1007/978-1-4939-7253-1_13
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Publisher Name: Humana Press, New York, NY
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