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Updated: Nov 7, 2025
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Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles
Published on: March 28, 2021
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Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking
Sobha Karuthedom George1, Lucia Lauková1, René Weiss1
1Center for Biomedical Technology, Department for Biomedical Research, Danube University Krems, 3500 Krems, Austria.
International Journal of Molecular Sciences
|April 30, 2021
Summary
Standardizing flow cytometry for extracellular vesicle (EV) characterization is crucial. This study reveals instrument differences significantly impact EV counts, emphasizing the need to report detected EV size ranges for comparable results.
Area of Science:
- Biotechnology
- Nanomedicine
- Cell Biology
Background:
- Extracellular vesicles (EVs) are vital for intercellular communication, with growing interest in their characterization.
- Flow cytometry is a key technique for EV analysis due to its high-throughput, multi-parameter, and single-particle capabilities.
- Current challenges in EV research include a lack of standardized protocols and instrument comparability, hindering data interpretation.
Purpose of the Study:
- To compare EV quantification using different flow cytometers and nanoparticle tracking analysis (NTA).
- To assess the impact of instrument detection limits on EV counts and phosphatidylserine exposure measurements.
- To highlight the necessity of reporting EV size ranges for improved inter-study comparability.
Main Methods:
- Quantification of platelet-derived EVs using Gallios and CytoFLEX LX flow cytometers and NTA.
- Identification of phosphatidylserine-exposing EVs via lactadherin (LA) labeling.
- Instrument calibration using silica-based fluorescent beads to determine detection limits.
Main Results:
- CytoFLEX LX exhibited a lower detection limit (150 nm) compared to Gallios (300 nm).
- CytoFLEX LX yielded significantly higher EV counts (40-fold) and LA+CD41+ EV counts (13-fold) than Gallios.
- Fluorescence NTA indicated only 9.5% of detected vesicles exposed phosphatidylserine, showing good agreement with LA+ EV counts from CytoFLEX LX.
Conclusions:
- Instrument choice and detection limits critically influence EV concentration measurements.
- Standardization requires reporting the size range of detected EVs alongside concentration data.
- Consistent reporting of EV size ranges is essential for robust and comparable research findings across studies.
