Application Note

Use of Fluorescence Nanoparticle Tracking Analysis as a tool for quality assessment of EVs


Proper storage of extracellular vesicles (EV) and standardized conditions are key factors and prerequisites for the stability of clinical samples from biobanks and the production of consistent EV batches for therapeutic use [1-2]. Loss of EV concentration due to improper storage conditions is accompanied by their reduced activity and physical integrity. As a result, surface proteins used for biochemical phenotyping may be degraded or have limited functionality [3-6].

Keywords: storage conditions, quality assessment, EV therapy, extracellular vesicles, EVs, F-NTA, fluorescence nanoparticle tracking analysis

Figure 1.: Fluorescence labeling of CD63 dTomato expressing EVs 0, 3, 6 and 10 days after storage at 25°C.

In this experiment HEK293T derived EVs overexpressing CD63 dTomato were simultaneous fluorescently labeled with CD63 APC and CD81 BV421. Labeling and fluorescence NTA (F-NTA) measurements were done immediately after the sample was thawed. Additional aliquots were stored at 25°C for 3, 6, and 10 days after thawing, followed by labeling and F-NTA analysis.

Within the 10 day experimental timeframe, a substantial reduction of EV concentration was observed in F-NTA via CD63 detection (Fig. 2). The labeling efficiency of CD63 APC compared with intrinsic CD63 dTomato labeling was, for any given day, usually near 100%. The stepwise reduction in CD63 dTomato and CD63 APC concentrations are nearly identical over the observed time intervals. These results indicate reliability in the experiments as well as compatibility between CD63 dTomato and further labeling with CD63 APC antibody. However, CD81 BV421 did not show any reduction over time.

Figure 2.: Significant reduction in EV concentration over 10 days of storage at 25°C; analyzed by scatter and F-NTA
Figure 3.: No significant change in EV size over the 10 days of storage at 25°C; analyzed by scatter and F-NTA.

A significant change in size, which is sometimes measured after thawing, was not observed, and is likely dependent on the membrane properties such as rigidity/fluidity or protein content of the EVs. However, it can be assumed that a constant degradation of vesicles, over our storage time intervals, was responsible for the reduction in EV counts measured via scatter and F-NTA.


The results show that storage of EVs at 25°C results in a constant and ongoing decrease in particle count in scatter NTA as well as a reduction in CD63 expressing vesicles, indicating that the observed decrease in the overall particle number is caused by a degradation of the vesicles.

Interestingly, the concentration of CD81 expressing vesicles remains stable over time, giving a hint that this subpopulation might be more stable under the given storage conditions.

Concentration measurements via scatter and F-NTA can provide a quick and reliable quality assessment. Size, concentration and fluorescence can be quickly measured on one EV sample in a single run on the ZetaView®.


1. Théry et al.:
Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
J Extracell Vesicles 2018 Nov 23; 7(1):1535750

2. Witwer et al.:
Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
Journal of Extracellular Vesicles 2013; 2:(1),

3. Bosch et al.:
Trehalose prevents aggregation of exosomes and cryodamage.
Scientific Reports 2016; 6, 36162

4. Lener et al.:
Applying extracellular vesicles based therapeutics in clinical trials – an ISEV position paper.
Journal of Extracellular Vesicles 2015, 4, 30087.

5. Mateescu et al.:
Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper.
Journal of Extracellular Vesicles 2017, 6(1),1286095.

6. Görgens et al.:
Identification of storage conditions stabilizing extracellular vesicles preparations.
Journal of Extracellular Vesicles 2022, 11:e12238.


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Copyright: © May 2024 Particle Metrix. This publication or parts thereof may not be copied or distributed without our express written permission..

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