Extracellular Vesicle Flow Cytometry Analysis and Standardization

Welsh, Joshua; Holloway, Judith A.; Wilkinson, James S.; Englyst, Nicola A.

doi:10.3389/fcell.2017.00078

Cited by 106 publications

(98 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Specific protein markers could be identified utilizing immunogold on TEM sections. This would allow flow cytometry of placental homogenates in order to identify and purify these stromal macrovesicles (Welsh et al 2019). Raman spectroscopy would be an in situ way of identifying whether the stromal macrovesicles contain lipid (Devitt et al 2018).…”

Section: Discussionmentioning

confidence: 99%

Human placental villi contain stromal macrovesicles associated with networks of stellate cells

et al. 2019

View full text Add to dashboard Cite

Placental function is essential for fetal development and establishing the foundations for lifelong health. The placental villous stroma is a connective tissue layer that supports the fetal capillaries and villous trophoblast. All the nutrients that cross the placenta must also cross the stroma, and yet little is known about this region. This study uses high‐resolution three‐dimensional imaging to explore the structural complexity of this region within the placental villi. Serial block‐face scanning electron microscopy and confocal microscopy were used to image the placental villous stroma in three‐dimensions. Transmission electron microscopy (TEM) was used to generate high resolution two‐dimensional images. Stereological approaches were used to quantify volumes of stromal constituents. Three‐dimensional imaging identified stromal extracellular vesicles, which constituted 3.9% of the villous stromal volume. These stromal extracellular vesicles were ovoid in shape, had a median length of 2750 nm (range 350–7730 nm) and TEM imaging confirmed that they were bounded by a lipid bilayer. Fifty‐nine per cent of extracellular vesicles were in contact with a fibroblast‐like stellate cell and these vesicles were significantly larger than those where no contact was observed. These stellate cells formed local networks with adherent junctions observed at contact points. This study demonstrates that the villous stroma contains extracellular macrovesicles which are considerably larger than any previously described in tissue or plasma. The size and abundance of these macrovesicles in the villous stroma highlight the diversity of extracellular vesicle biology and their roles within connective tissues.

show abstract

Section: Discussionmentioning

confidence: 99%

Human placental villi contain stromal macrovesicles associated with networks of stellate cells

et al. 2019

View full text Add to dashboard Cite

show abstract

“…In recent years, the progress attained in the field of EV research has drastically changed methodological approaches based on multiparameter FCM. Despite being one of the most promising techniques for EV characterization, its successful use implies accurate setup of the instrument (Welsh et al, 2017).…”

Section: Flow Cytometry Setup Acquisition and Analysismentioning

confidence: 99%

“…Therefore, many attempts have been made to ameliorate both isolation techniques and characterization methods. Advanced flow cytometry (FCM) is one of the most promising approaches, as it can be used to analyze a large range of particle diameters <1 μm; in contrast, conventional FCM is useful for appropriately discriminating only larger particles (e.g., platelets) (Van Der Vlist, Nolte, Stoorvogel, Arkesteijn, & Wauben, 2012;Welsh, Holloway, Wilkinson, & Englyst, 2017).…”

Section: Introductionmentioning

confidence: 99%

Isolation and Flow Cytometry Characterization of Extracellular‐Vesicle Subpopulations Derived from Human Mesenchymal Stromal Cells

Görgün

Reverberi

Rotta³

et al. 2019

CP Stem Cell Biology

View full text Add to dashboard Cite

This unit describes protocols for isolating subpopulations of extracellular vesicles (EVs) purified from human adipose tissue–derived mesenchymal stromal cells by density gradient centrifugation and for characterizing them by flow cytometry (FCM). Determining the optimal strategy for isolating EVs is a critical step toward retrieving the maximal amount while ensuring the recovery of different vesicular subtypes. The first protocol details density gradient centrifugation to isolate both exosomes and microvesicles. In the second protocol, characterization of EV subpopulations by FCM is depicted, taking advantage of non‐conventional modalities, in accordance with the latest technical indications. The procedures described here can be easily reproduced and can be employed regardless of the cell type used to obtain EVs. © 2019 by John Wiley & Sons, Inc.

show abstract

“…Its principle is based on light scattering and fluorescence emission of the particle, with fluorescent features derived from antibodies or dyes, upon illumination with a series of laser beams. The detected signals are then processed for analysis [250][251][252][253]. FC gives a statistically relevant and quantitative signal of fluorescence very quickly and allows sensitive detection.…”

Section: Live Imagingmentioning

confidence: 99%

Membrane Active Peptides and Their Biophysical Characterization

Avcı

Akbulut

Özkırımlı

2018

Preprint

View full text Add to dashboard Cite

In the last 20 years, an increasing number of studies have been reported on membrane active peptides, which exert their biological activity by interacting with the cell membrane either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. These peptides are attractive alternatives to currently used pharmaceuticals. Antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery currently in the drug pipeline suggest that these membrane active peptides will soon constitute a significant percentage of the drug market. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). AMPs are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus and fungi. CPPs are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity to some extent. Biophysical techniques to understand how they interact with the membrane have shed light on the peptide-membrane interaction at various levels of detail. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Advances in live imaging techniques have allowed examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provided clues on the peptide-lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.

show abstract

Extracellular Vesicle Flow Cytometry Analysis and Standardization

Cited by 106 publications

References 52 publications

Human placental villi contain stromal macrovesicles associated with networks of stellate cells

Human placental villi contain stromal macrovesicles associated with networks of stellate cells

Isolation and Flow Cytometry Characterization of Extracellular‐Vesicle Subpopulations Derived from Human Mesenchymal Stromal Cells

Membrane Active Peptides and Their Biophysical Characterization

Contact Info

Product

Resources

About