Quantification of Nanosized Extracellular Membrane Vesicles with Scanning Ion Occlusion Sensing

Vrij, Jeroen de; Maas, Sybren L. N.; Nispen, Malisa Van; Sena‐Esteves, Miguel; Limpens, Ronald W. A. L.; Koster, Abraham J.; Leenstra, Sieger; Lamfers, Martine L.M.; Broekman, Marike L. D.

doi:10.2217/nnm.12.173

Cited by 107 publications

(118 citation statements)

References 47 publications

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“…A 5 mm obscuration bar was placed in front of the FSC collection lens. FSC was measured with a collection angle of [15][16][17][18][19][20][21][22][23][24][25] (reduced wide-angle FSC; rw-FSC). We previously optimized the detection of FSC induced by nano-sized particles by using a larger (5 mm) obscuration bar, high numerical aperture and long working distance lens, and by reconstruction of the BD small particle detector (11).…”

Section: High-resolution Flow Cytometric Analysis Of Submicron-sized mentioning

confidence: 99%

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Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

Kormelink

Arkesteijn

Nauwelaers³

et al. 2015

Cytometry Pt A

170

180

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Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for highthroughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a highresolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible. V C 2015 International Society for Advancement of Cytometry Key terms extracellular vesicle; exosome; microvesicle; microparticle; high-resolution flow cytometry; characterization; sorting; coincidence; swarm; liposome EXTRACELLULAR vesicles (EVs) are small membrane-enclosed vesicles released by cells either by outward budding from the plasma membrane or by the fusion of multivesicular bodies with the plasma membrane resulting in the release of intracellular stored vesicles (1). The release of EVs and their content, i.e., proteins, lipids and RNAs, is tightly regulated and varies not only between different cell types but also depends on the physiological state of the producing cell (2-4). Consequently, EV release is very dynamic and the EV population is very heterogeneous. EVs can function in an autocrine or paracrine fashion, but can also enter the circulatory system and act at distant sites. Hence EVs are present in body fluids like blood, milk, urin...

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Section: High-resolution Flow Cytometric Analysis Of Submicron-sized mentioning

confidence: 99%

“…3H). Notably, other frequently used methods for EV analysis, e.g., NTA and tRPS, require sample concentrations that lead to swarm in flow cytometry (21,22).…”

mentioning

confidence: 99%

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

Kormelink

Arkesteijn

Nauwelaers³

et al. 2015

Cytometry Pt A

170

180

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show abstract

“…Other authors have trialled spiking biological fluids with polystyrene beads of known size and concentration to improve EV measurement accuracy [19,20]. For NTA, a similar approach using silica beads would be less useful as the scatter mode does not discriminate between particles of similar size.…”

Section: Variability In Measurements and Techniquesmentioning

confidence: 99%

Challenges in Analysis of Circulating Extracellular Vesicles in Human Plasma Using Nanotracking and Tunable Resistive Pulse Sensing

Enjeti¹,

Ariyarajah²,

Warwick³

et al. 2017

J Nanomed Nanotechnol

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“…As a result, there have been a surge in the number of diagnostic modalities to study EVs. Over the last few years, a number of studies have employed TRPS as a diagnostic tool to characterise EVs [48][49][50][51][52].…”

Section: Trps In Extracellular Vesicles Analysismentioning

confidence: 99%

“…de Vrij et al [48] investigated the ability of TRPS to quantify non-purified EVs in various body fluids including urine, blood plasma and pleural fluid and culture supernatants. They found that, in all the samples studied, more than 95% of particles were in the range of 150-400nm.…”

Section: Trps In Extracellular Vesicles Analysismentioning

confidence: 99%

Tunable Resistive Pulse Sensing: Potential Applications in Nanomedicine

Sivakumaran

Platt

2016

Nanomedicine (Lond.)

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An accurate characterisation of nanomaterials used in clinical diagnosis and therapeutics is of paramount importance to realise the full potential of nanotechnology in medicine and to avoid unexpected and potentially harmful toxic effects due to these materials. A number of technical modalities are currently in use to study the physical, chemical and biological properties of nanomaterials but they all have advantages and disadvantages. In this review, we discuss the potential of a relative newcomer, Tunable Resistive Pulse Sensing (TRPS), for the characterisation of nanomaterials and its applications in nanodiagnostics.

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Quantification of Nanosized Extracellular Membrane Vesicles with Scanning Ion Occlusion Sensing

Abstract: Scanning ion occlusion sensing technology enables the quantification of MVs in biological samples without the requirement of MV isolation and/or labeling. This offers a highly valuable addition to the currently used repertoire of MV quantification methods.

Cited by 107 publications

References 47 publications

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

Challenges in Analysis of Circulating Extracellular Vesicles in Human Plasma Using Nanotracking and Tunable Resistive Pulse Sensing

Tunable Resistive Pulse Sensing: Potential Applications in Nanomedicine

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