Analysis of Extracellular Vesicles Using Fluorescence Nanoparticle Tracking Analysis

  • Pauline Carnell-Morris
  • Dionne Tannetta
  • Agnieszka Siupa
  • Patrick Hole
  • Rebecca DragovicEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1660)


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.

Key words

Extracellular vesicles Exosomes Microvesicles Fluorescence nanoparticle tracking analysis Quantum dots 



This work was supported by MRC programme grant MR/J003360/1.


  1. 1.
    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 PubMedGoogle Scholar
  2. 2.
    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 CrossRefPubMedGoogle Scholar
  3. 3.
    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
  4. 4.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    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
  7. 7.
    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 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Pauline Carnell-Morris
    • 1
  • Dionne Tannetta
    • 2
  • Agnieszka Siupa
    • 1
  • Patrick Hole
    • 1
  • Rebecca Dragovic
    • 3
    Email author
  1. 1.Malvern Instruments LtdAmesburyUK
  2. 2.Department of Food and Nutritional SciencesUniversity of ReadingReadingUK
  3. 3.Nuffield Department of Obstetrics & GynaecologyUniversity of OxfordOxfordUK

Personalised recommendations