Absolute sizing and label-free identification of extracellular vesicles by flow cytometry

Pol, Edwin van der; Rond, Leonie de; Coumans, F.A.W.; Gool, Elmar L.; Böing, Anita N.; Sturk, Auguste; Nieuwland, Rienk; Leeuwen, Ton G. van

doi:10.1016/j.nano.2017.12.012

Cited by 109 publications

(143 citation statements)

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“…The Mie scattering theory that describes the scattering properties of spherical nanoparticles with a size comparable to the wavelength used to probe the particles (13) is however far more complex than the simple radius-squared relation between the size of liposomes and their corresponding fluorescence intensity (4,25). While more sensitive SSC and/or FSC detection setups than the FACSAria setup used in this study may provide scattering output useful for vesicle size determination (12,13), we find that the SSC resolution to be limited for small (30-300 nm) liposomes. Hence, by sorting several fractions according to the SSC of different sizes of nanoparticles, well-defined size bins can be defined and used to provide a size distribution of nanoparticles based on the flow cytometric scattering readout, similar to the FANS procedure presented herein.…”

Section: Discussionmentioning

confidence: 99%

“…In principle, liposomes could be used as calibrators for EV size determination due to the structural similarity between liposomes and EVs (both are core-shell structure), instead of using solid plastic and silica beads that are traditionally used as EV size calibrators. Recent work (12) shows that by taking the ratio between SSC and FSC (they used a high scatter sensitivity flow cytometer) in combination with Mie scattering theory, EV sizes in the range from 200 to 500 nm could be deduced. Yet, EVs are a very heterogeneous population of biological nanoparticles when it comes to size and composition and therefore also refractive indexes.…”

Section: The Potential Use Of Flow-activated Nanoparticle-sorted Calimentioning

confidence: 99%

“…First, the detection sensitivity of conventional flow cytometers has to be improved. A theoretical approach based on Mie scattering theory has also been applied to assign sizes to EVs based on their scattering intensity (12,13). These improvements were obtained by increasing the exposure time (by decreasing the flow rate of the sheath fluid), increasing the laser power, and improving the system optics.…”

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confidence: 99%

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Unique Calibrators Derived from Fluorescence‐Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations

et al. 2019

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The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles.TO be able to analyze multiple parameters of individual nanoparticles, including their size, in a high-throughput manner has long been a goal in nanoparticle research. Traditional tools for nanoparticle characterization are often limited by a low throughput, such as microscopy-based methods, or restricted to single-property measurements using ensemble-based methods. The potential problem with employing bulk absorption and emission spectroscopy-based methods or dynamic light small-angle X-ray or neutron scattering techniques is that the ensemble average information they provide

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Section: Discussionmentioning

confidence: 99%

Section: The Potential Use Of Flow-activated Nanoparticle-sorted Calimentioning

confidence: 99%

mentioning

confidence: 99%

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Unique Calibrators Derived from Fluorescence‐Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations

et al. 2019

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“…The measurement data are analyzed by a standalone software package that automatically recognizes the bead populations and fits a Mie model to derive the parameters describing the optical configuration of the used FCM. Starting from the manufacturer's specification for each FCM, these optical parameters are fitted to different values for each FCM, accounting for differences in both design and FCM alignment; see for more details. With the assumption of an RI of 1.40 for EVs , the scatter–diameter relationship for EVs is estimated.…”

Section: Methodsmentioning

confidence: 99%

Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

Pol

Sturk

Leeuwen

et al. 2018

Journal of Thrombosis and Haemostasis

136

137

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Background Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights into the diameter of detected EVs. Objectives To evaluate gates based on the estimated diameter of EVs instead of beads. Methods A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates in order to measure the concentration of CD61-phycoerythrin-positive platelet EVs. Results Of the 46 evaluated FCMs, 21 FCMs detected the 600-1200-nm EV diameter gate. The 1200-3000-nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL min differed six-fold in measured flow rates between instruments. Conclusions EV diameter gates improve the interlaboratory variability as compared with previous approaches. Of the evaluated FCMs, 24% could not detect 400-nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity.

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“…However, these imaging-based analyses are low-throughput and not easily amenable to determination of EV contents. Adaptation of flow cytometric protocols has allowed for measurement of proteins in individual EVs and identification of separate EV populations [3*]. Poor signal-to-noise ratios remain a problem in these measurements, limiting its ability to identify low abundance proteins.…”

Section: Introductionmentioning

confidence: 99%

Extracellular vesicles in cardiovascular homeostasis and disease

Hutcheson

Aikawa

2018

Current Opinion in Cardiology

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Purpose of review Extracellular vesicles (EVs) have emerged as one of the most important means through which cells interact with each other and the extracellular environment, but EV research remains challenging due to their small size and limited amount of material required for traditional molecular biology assays. The advent of new technologies and standards in the field, however, have led to increased mechanistic insight into EV function. Herein, the latest studies on the role of EVs in cardiovascular physiology and pathology are discussed. Recent findings EVs help control cardiovascular homeostasis and remodeling by mediating communication between cells and directing alterations in the extracellular matrix to respond to changes in the environment. The message carried from the parent cell to extracellular space can be intended for both local (within the same tissue) and distal (downstream of blood flow) targets. Pathological cargo loaded within EVs could further result in various diseases. On the other hand, new studies indicate that injection of EVs obtained from cultured cells into diseased tissues can promote restoration of normal tissue function. Summary EVs are an integral part of cell and tissue function, and harnessing the properties inherent to EVs may provide a therapeutic strategy to promote tissue regeneration.

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Absolute sizing and label-free identification of extracellular vesicles by flow cytometry

Cited by 109 publications

References 40 publications

Unique Calibrators Derived from Fluorescence‐Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations

Unique Calibrators Derived from Fluorescence‐Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations

Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

Extracellular vesicles in cardiovascular homeostasis and disease

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