网络研讨会

EV Characterization through Nanoscale Flow Cytometry

九月 11, 2025

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Learning objectives:

Participants will understand how to calibrate the CytoFLEX nano instrument to analyze small vesicles under 300 nm. In addition, they will be able to appreciate the differences and sensitivity of the two violet scatter channels.

Given the challenges in determining the collection gate for EVs, participants will observe how the use of reference standards—such as exosomes derived from HEK cells expressing GFP—makes it easier to define both positive and negative collection gates for vesicles.

An additional aspect that participants will learn is how to verify whether the EVs are composed of a phospholipid bilayer, and how to exclude single-layer vesicles by using ACO markers—electrolyte dyes that intercalate only when vesicles have a true phospholipid bilayer structure.

One ongoing debate that we aim to address is the determination of vesicle size using calibration beads. Participants will explore how polystyrene calibration beads work and how to use the FCMPASS software to calculate the size of EVs, based on the known refractive index of the beads. They will also observe how, by using nanoViS, it becomes easier to determine vesicle size through FCMPASS.

Of course, participants will also learn how to perform an experiment to detect circulating vesicles in plasma using this instrument, applying the strategies described above. Approaches or techniques that can also be applied to other CytoFLEX cytometers.

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Introduction: Extracellular Vesicles (EVs) are key mediators of intercellular communication, enclosed by a phospholipid bilayer and lack the ability to replicate. Characterization of EVs is essential to elucidate their cellular origins, molecular cargo, and functional roles in health and disease.  However, the nanoscale size and low refractive index (RI) present significant challenges for detection and size determination with conventional flow cytometers. Furthermore, the use of standard polystyrene beads for calibration complicates the analysis due to their significantly higher light-scattering properties compared to EVs. The CytoFLEX nano represents a new platform for EV evaluation. The instrument is equipped with a multiple-scatter laser which increases the sensitivity for the detection of small EVs. By combining scatter-based detection with fluorescence antibodies labelling, size, concentration, and expression of EVs surface markers can be measured simultaneously, providing a more accurate and sensitive approach to EVs analysis.
 
Aim: The aim of this study was to calibrate the CytoFLEX nano flow cytometer for the detection and analysis of small EVs with diameters less than 300 nm. For this purpose, the Cellarcus Vesicle Analysis Kit (vFC™) and AcoDyes™ were employed. Following calibration, plasma-derived EVs were analysed. Additionally, nanoViS polystyrene beads were used in combination with FCMPASS software to estimate the size distribution of EVs.
 
Materials and Methods: The CytoFLEX nano is equipped with multiple scatter and fluorescence detection channels. Calibration and standardization were performed using the vFC™ Kit, which includes lipid-based vesicle standards that closely mimic the refractive index (RI) of native EVs, along with two fluorescently labelled EV reference particles. AcoDyes™ probes, a class of conjugated oligo-electrolytes, were used for membrane labelling. These dyes are able to spontaneously intercalate into the lipid bilayer of EV membranes.
To estimate the precise size of EVs, nanoViS Nanoscale Sizing Standards, which are polystyrene beads spanning the nanoscale detection range from 40 to 1000 nm, were used. These standards are ideal for determining the cytometer’s scatter dynamic range and establishing calibration curves.
As a positive control, fluorescent recombinant exosomes derived from human HEK293 cells, expressing green fluorescent protein (GFP) on their membrane surface, were used. These standard exosomes diplayed a typical size distribution from 30 to 300 nm, with a peak between 100 and 150 nm.
After calibration, plasma samples from healthy donors were stained with Calcein-AM, Aco-600, CD41 and CD62P-selectin. Finally, FCMPASS software was used to convert the light scattering data into nanometric size units and to determine the size distribution of EVs.
 
Results: The vFC™ kit was used to calibrate the light scatter of the instrument and to measure the concentration and size of EVs using the nanoCal sizing standards. The gate collection of positive and negative EVs was designed using the unstained lyophilized reference EVs as negative and as negative controls and the stained ones (EVs-520, EVs-615) as positive controls. A mixture of nanoViS multi-size polystyrene beads—including sizes of 44 nm, 80 nm, 100 nm, 144 nm, 300 nm, 600 nm, and 1 µm—with a defined refractive index was used to calibrate the instrument for EVs size analysis. GFP-expressing exosome standard EVs served as a positive control for size estimation, confirming a size range of approximately 30 to 300 nm, as expected.
 
After instrument calibration, plasma platelet-derived EVs were identified. Aco-600 staining was performed to determine the phospholipid bilayer integrity of the EVs. Calcein-AM positivity was used to assess the vitality of the EVs, while the markers CD62P and CD41 confirmed their platelet origin. Using the nanoViS beads and FCMPASS software, the size of plasma EVs was calculated, revealing a size distribution ranging from 40 to 300 nm and demonstrating the instrument’s ability to resolve nanoscale heterogeneity.
 
Conclusions: The CytoFLEX nano cytometer, featuring enhanced sensitivity of the VSSC1 detector, enables the detection of particles as small as 40 nm. When combined with the vFC™ Cellarcus Vesicle Analysis Kit and AcoDyes™, this platform provides a powerful tool for detailed nanoscale analysis of EVs, including their size, molecular composition, and surface properties. Furthermore, the use of nanoViS polystyrene beads in conjunction with FCMPASS software allows accurate determination of the size distribution of circulating EVs. These advancements contribute to the development of standardized protocols, addressing critical challenges in EVs characterization and supporting their future application in diagnostic and therapeutic settings.
Learning objectives:
Participants will understand how to calibrate the CytoFLEX nano instrument to analyze small vesicles under 300 nm. In addition, they will be able to appreciate the differences and sensitivity of the two violet scatter channels.
 
Given the challenges in determining the collection gate for EVs, participants will observe how the use of reference standards—such as exosomes derived from HEK cells expressing GFP—makes it easier to define both positive and negative collection gates for vesicles.
 
An additional aspect that participants will learn is how to verify whether the EVs are composed of a phospholipid bilayer, and how to exclude single-layer vesicles by using ACO markers—electrolyte dyes that intercalate only when vesicles have a true phospholipid bilayer structure.
 
One ongoing debate that we aim to address is the determination of vesicle size using calibration beads. Participants will explore how polystyrene calibration beads work and how to use the FCMPASS software to calculate the size of EVs, based on the known refractive index of the beads. They will also observe how, by using nanoViS, it becomes easier to determine vesicle size through FCMPASS.
 
Of course, participants will also learn how to perform an experiment to detect circulating vesicles in plasma using this instrument, applying the strategies described above. Approaches or techniques that can also be applied to other CytoFLEX cytometers.
For Research Use Only. Not for use in diagnostic procedures.
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