Physical Characterization of Mouse Deep Vein Thrombosis Derived Microparticles by Differential Filtration with Nanopore Filters

Peramo, Antonio; Díaz, José

doi:10.3390/membranes2010001

Cited by 10 publications

(8 citation statements)

References 34 publications

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“…We suppose that not‐identified particles correspond to a larger MPs aggregates or some other plasma constituents. In particular, such aggregates were previously detected by other methods, such as confocal and electron microscopy . The FSC versus true MP diameters ( d or d ev ) are separately shown in the inset in Figure A.…”

Section: Resultsmentioning

confidence: 54%

Super‐resolved calibration‐free flow cytometric characterization of platelets and cell‐derived microparticles in platelet‐rich plasma

et al. 2015

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Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in plateletrich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population. V C 2015 International Society for Advancement of Cytometry

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Section: Resultsmentioning

confidence: 54%

Super‐resolved calibration‐free flow cytometric characterization of platelets and cell‐derived microparticles in platelet‐rich plasma

et al. 2015

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“…In order to analyze EVs, researchers have utilized various methods, including transmission electron microscopy 17 , scanning electron microscopy 18,19 , atomic force microscopy, dynamic light scattering 20,21 and western blotting 22,23 . While FCM is the method of choice for many researchers 9,[24][25][26] due to its high throughput capabilities, analysis of EVs using FCM has been notoriously difficult due to their size and lack of discrete positive populations [27][28][29][30][31][32] .…”

Section: Introductionmentioning

confidence: 99%

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Inglis

Norris

Danesh

2015

JoVE

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Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal-to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes. Video LinkThe video component of this article can be found at

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“…A number of methods have been used to analyze EVs, including scanning electron microscopy (39,40), transmission electron microscopy (TEM) (41), atomic force microscopy and dynamic light scattering (42)(43)(44)(45), and western blotting (46,47). Clinically, flow cytometry (FCM) is the most commonly used method for analyzing EVs in blood (5,(48)(49)(50); however, accurate characterization of EVs remains challenging.…”

mentioning

confidence: 99%

Techniques to improve detection and analysis of extracellular vesicles using flow cytometry

et al. 2015

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Extracellular vesicles (EVs) range in size from 50 nm to 1 μm. Flow cytometry (FCM) is the most commonly used method for analyzing EVs; however, accurate characterization of EVs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques that are especially well-suited for analyzing EVs from a high volume of clinical samples. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-EV events. In addition, we show that lysed samples are a useful alternative to isotypes for setting gates to exclude background fluorescence. To reduce background we developed an approach using filters to “wash” samples post-staining thus providing a faster alternative to ultracentrifugation and sucrose gradient fractionation. In conclusion, use of these optimized techniques enhances the accuracy and efficiency of EV detection using FCM.

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Physical Characterization of Mouse Deep Vein Thrombosis Derived Microparticles by Differential Filtration with Nanopore Filters

Cited by 10 publications

References 34 publications

Super‐resolved calibration‐free flow cytometric characterization of platelets and cell‐derived microparticles in platelet‐rich plasma

Super‐resolved calibration‐free flow cytometric characterization of platelets and cell‐derived microparticles in platelet‐rich plasma

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Techniques to improve detection and analysis of extracellular vesicles using flow cytometry

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