The more the better – determining the optimal range when performing single-vesicle phenotyping

2021 ◽  
Vol 1 (1) ◽  
pp. 26-33
Author(s):  
María Gómez-Serrano ◽  
Christian Preußer ◽  
Kathrin Stelter ◽  
Elke Pogge von Strandmann
Keyword(s):  
Extracellular Vesicles ◽  
Particle Number ◽  
Experiment Design ◽  
Surface Markers ◽  
Optimal Range ◽  
Design And Optimization ◽  
Highly Sensitive ◽  
Single Vesicle

The characterization of extracellular vesicles (EVs) has evolved rapidly in recent years due to advances in straightforward technologies. Based on these more sensitive methods, it is now possible to describe EV populations in their entirety more precisely. However, these applications require an equivalently delicate experiment design and optimization steps to draw valid conclusions in the end. One of these methods is represented by the highly sensitive nanoflow cytometry (nFCM), by which particles can be analyzed not only on their size (< 40 nm) and concentration but also concerning surface markers. In this work, we addressed some of the potential caveats of this method, especially when characterizing particles with fluorescently labelled antibodies. In particular, we show, when using low particle concentrations, which are inevitably encountered when working with EVs, the characterization of surface markers is prone to significantly varying. We hypothesized that these technical limitations could respond to the stickiness of EVs and should be properly counteracted. As a reference, we strongly recommend performing particle number-based comparisons with at least 109 particles as staining input in nFCM analyses. Moreover, we provided representative particle-number based immunoblotting results, underlying the significance of this parameter as a normalizer in future EV research.

scholarly journals The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2272
Author(s):  
Hussein Kaddour ◽  
Malik Tranquille ◽  
Chioma M. Okeoma
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Target Cells ◽  
Purification And Characterization ◽  
Extracellular Milieu ◽  
Exclusion Chromatography ◽  
Single Vesicle

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

scholarly journals Molecular Characterization of Temozolomide-Treated and Non Temozolomide-Treated Glioblastoma Cells Released Extracellular Vesicles and Their Role in the Macrophage Response

2020 ◽  
Vol 21 (21) ◽  
pp. 8353
Author(s):  
Elisa Panzarini ◽  
Stefano Tacconi ◽  
Elisabetta Carata ◽  
Stefania Mariano ◽  
Ada Maria Tata ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Extracellular Vesicles ◽  
Cell Activation ◽  
Cell Communication ◽  
Molecular Profile ◽  
Surface Markers ◽  
Macrophage Response ◽  
A Cell ◽  
Potential Biomarkers

Extracellular vesicles (EVs) are widely investigated in glioblastoma multiforme (GBM) for their involvement in regulating GBM pathobiology as well as for their use as potential biomarkers. EVs, through cell-to-cell communication, can deliver proteins, nucleic acids, and lipids that are able to reprogram tumor-associated macrophages (TAMs). This research is aimed to concentrate, characterize, and identify molecular markers of EVs subtypes released by temozolomide (TMZ)-treated and non TMZ-treated four diverse GBM cells. Morphology, size distribution, and quantity of small (sEVs) and large (lEVs) vesicles were analyzed by cryo-TEM. Quality and quantity of EVs surface markers were evaluated, having been obtained by Western blotting. GBM cells shed a large amount of EVs, showing a cell line dependent molecular profile A comparative analysis distinguished sEVs and lEVs released by temozolomide (TMZ)-treated and non TMZ-treated GBM cells on the basis of quantity, size and markers expression. Finally, the GBM-derived sEVs and lEVs, irrespective of TMZ treatment, when challenged with macrophages, modulated cell activation toward a tendentially M2b-like phenotype.

scholarly journals The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

2021 ◽  
Author(s):  
Hussein Kaddour ◽  
Malik Tranquille ◽  
Chioma M. Okeoma
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Target Cells ◽  
Purification And Characterization ◽  
Extracellular Milieu ◽  
Exclusion Chromatography ◽  
Single Vesicle

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

scholarly journals Circulating extracellular vesicles of steroid sensitive nephrotic syndrome patients have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes

2021 ◽  
Author(s):  
Fehime Kara Eroglu ◽  
Volkan Yazar ◽  
Ulku Guler ◽  
Muzaffer Yıldırım ◽  
Tugce Yildirim ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Extracellular Vesicles ◽  
Particle Number ◽  
Biologically Active ◽  
Soluble Mediator ◽  
Steroid Sensitive Nephrotic Syndrome ◽  
Steroid Sensitive

Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (SSNS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with SSNS in relapse and remission, 10 healthy controls and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry and western blotting. The major proteins from the plasma EVs were identified via mass spectrometry. A Gene Ontology classification analysis and integuinity pathway analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and the particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively express proteins which involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared to remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho p38 (p-p38) and decreased levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the nephrotic syndrome phenotype in podocytes in vitro.

Synthesis and 31 P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies

2002 ◽  
Vol 10 (5) ◽  
pp. 1451-1458 ◽  
Author(s):  
Sophie Martel ◽  
Jean-Louis Clément ◽  
Agnès Muller ◽  
Marcel Culcasi ◽  
Sylvia Pietri
Keyword(s):  
Acidic Ph ◽  
Highly Sensitive ◽  
Nmr Characterization ◽  
Low Toxic ◽  
Ph Probes

scholarly journals Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 139
Author(s):  
Johanna Detzner ◽  
Elisabeth Krojnewski ◽  
Gottfried Pohlentz ◽  
Daniel Steil ◽  
Hans-Ulrich Humpf ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Saturated Fatty Acids ◽  
Human Kidney ◽  
Enterohemorrhagic Escherichia Coli ◽  
Highly Sensitive ◽  
Liquid Ordered ◽  
Uremic Syndrome

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic–uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

scholarly journals Terahertz characterization of two-dimensional low-conductive layers enabled by metal gratings

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Prashanth Gopalan ◽  
Yunshan Wang ◽  
Berardi Sensale-Rodriguez
Keyword(s):  
Optical Transmission ◽  
Terahertz Spectroscopy ◽  
Design Guidelines ◽  
Two Dimensional ◽  
Highly Sensitive ◽  
Transmission Measurements ◽  
Metal Gratings ◽  
Low Sensitivity ◽  
Non Destructive

AbstractWhile terahertz spectroscopy can provide valuable information regarding the charge transport properties in semiconductors, its application for the characterization of low-conductive two-dimensional layers, i.e., σs <  < 1 mS, remains elusive. This is primarily due to the low sensitivity of direct transmission measurements to such small sheet conductivity levels. In this work, we discuss harnessing the extraordinary optical transmission through gratings consisting of metallic stripes to characterize such low-conductive two-dimensional layers. We analyze the geometric tradeoffs in these structures and provide physical insights, ultimately leading to general design guidelines for experiments enabling non-contact, non-destructive, highly sensitive characterization of such layers.

scholarly journals Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles

2021 ◽  
Author(s):  
Guillermo Bordanaba-Florit ◽  
Félix Royo ◽  
Sergei G. Kruglik ◽  
Juan M. Falcón-Pérez
Keyword(s):  
Extracellular Vesicles ◽  
Single Vesicle
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