scholarly journals Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently

2021 ◽  
Vol 22 (22) ◽  
pp. 12436
Author(s):  
Marvin Droste ◽  
Tobias Tertel ◽  
Stefanie Jeruschke ◽  
Robin Dittrich ◽  
Evangelia Kontopoulou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Storage Temperature ◽  
Extracellular Vesicle ◽  
Urine Samples ◽  
Size Exclusion ◽  
Preparation Technique ◽  
Nanoparticle Tracking Analysis ◽  
Preparation Methods ◽  
Imaging Flow Cytometry

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.

scholarly journals Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

2021 ◽  
Author(s):  
Marvin Droste ◽  
Tobias Tertel ◽  
Stefanie Jeruschke ◽  
Robin Dittrich ◽  
Evangelia Kontopoulou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Method Comparison ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Western Blots ◽  
Preparation Methods ◽  
Imaging Flow Cytometry ◽  
Particle Yield ◽  
Exclusion Chromatography ◽  
Ultra Filtration

Extracellular vesicles (EVs) from several body fluids, including urine, appear as promising biomarkers. Within the last decade, numerous groups have compared the efficacy of EV preparation protocols. Frequently, the efficacy of EV preparation methods is judged by the recovery of particles as estimated by conventional nanoparticle tracking analysis (NTA) or other particle quantification devices. Here, at the example of different urinary EV (uEV) preparation methods, we determined the particle yield in obtained samples with conventional NTA, analyzed their EV content by imaging flow cytometry (IFCM) and quantified the intensity of TSG101 and the contaminant protein uromodulin (UMOD) in Western blots. Our results demonstrate a correlation among CD9-positive objects detected by IFCM and TSG101 Western blot intensities, while particle numbers as determined by NTA correlated with the amount of UMOD. Consequently, our results question the reliability of conventional NTA analyses for identifying the optimal EV preparation method. Here, in our method comparison, a combination of size exclusion chromatography followed by ultra-filtration showed the highest CD9-positive object and TSG101 protein recovery, and in relation to the number of CD9-positive objects, the lowest amount of UMOD contamination.

scholarly journals Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

2020 ◽  
Author(s):  
Brian Jurgielewicz ◽  
Yao Yao ◽  
Steven L. Stice
Keyword(s):  
Flow Cytometry ◽  
Genetic Material ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Human Neural Stem Cells ◽  
Imaging Flow Cytometry ◽  
Gene Therapy Vectors ◽  
Time Variables

Abstract Background : Extracellular vesicles (EVs) are nanosized vesicles naturally secreted from cells responsible for intercellular communication and delivery of proteins, lipids, and other genetic material. Ultimately, EVs could provide innate therapeutic contents and loaded therapeutic payloads such as small molecules and gene therapy vectors to recipient cells. However, comparative kinetic measures that can be used to quantify and ultimately optimize delivery and uptake of EV payloads are lacking. We investigated both dose and time effects on EV uptake and evaluated the potential specificity of EV uptake to better understand the kinetics and uptake of human embryonic kidney (HEK293T) derived EVs. Results : Utilizing an imaging flow cytometry platform (IFC), HEK293T EV uptake was analyzed. HEK293T EV uptake was dose and time dependent with a minimum threshold dose of 6,000 EVs per cell at 4 hours of co-culture. HEK293T EV uptake was inhibited when co-cultured with recipient cells at 4°C or with pre-fixed recipient cells. By co-culturing HEK293T EVs with cell lines from various germ layers, HEK293T EVs were taken up at higher quantities by HEK293T cells. Lastly, human neural stem cells (hNSCs) internalized significantly more HEK293T EVs relative to mature neurons. Conclusions : Imaging flow cytometry is a quantitative, high throughput, and versatile platform to quantify the kinetics of EV uptake. Utilizing this platform, dose and time variables have been implicated to affect EV uptake measurements making standardization of in vitro and in vivo assays vital for the translation of EVs into the clinic. In this study, we quantified the selectivity of EV uptake between a variety of cell types in vitro and found that EVs were internalized at higher quantities by cells of the same origin. The characterization of HEK293T EV uptake in vitro, notably specificity, dose response, and kinetic assays should be used to help inform and develop EV based therapeutics.

scholarly journals Preparation of Nanoliposomes Incorporated Leishmania donovani Antigens

2016 ◽  
Vol 10 (2) ◽  
pp. 65-72
Author(s):  
Ali A. Taha ◽  
Rawaa Najim A. ◽  
Shaimaa yousif A.
Keyword(s):  
Fourier Transform ◽  
Infrared Spectroscopy ◽  
Electron Microscope ◽  
Leishmania Donovani ◽  
Storage Temperature ◽  
Entrapment Efficiency ◽  
Size Exclusion ◽  
Preparation Methods ◽  
The Stability ◽  
Scanning Electron

This study was designed to incorporate leishmania donovani antigens in nanoliposomes prepared by size exclusion (using Sephadex G25) and organic solvent (using Chloroform). Lipids mixture of 4Mm Phosphatidylcholine, 2.2mM Cholesterol and 0.55mM Phosphatidylethanolamine in a ratio of 7:2:1 was depended in two nanoliposome preparation methods. Physio-chemical characterizations of prepared nanoliposomes was performed by using Scanning Electron Microscope (SEM ), Fourier transform infrared spectroscopy (FTIR) and  Zeta Potential assays to determine the size, morphology, chemical active group and charge . Parasite reactivation was carried out when inoculated into RPMI and incubated at 23 ̊ C for 4 days. Soluble Leishmania Antigenes (SLAs) were extracted from the promastigotes ghost membrane after fourth passages of subculturing in SNB9. The extracted SLAs were entrapped in prepared. The percentage of nanoliposomes entrapment efficiency (EE) was 62 and 50 of SLAs for chloroform and Sephadex G25 methods, respectively. Moreover, stability of SLAs entrapped nanoliposomes at 4 and 37 ̊C, as storage temperature, was examined. The stability at 4 °C showed decreasing in EE to 32 and 16 %, while stability at 37 °C revealed decreasing in EE to 16 and 8 % within 12 days of storage for nanoliposomes prepared in both methods, respectively.

scholarly journals Efficient Neutrophil Activation Requires Two Simultaneous Activating Stimuli

2021 ◽  
Vol 22 (18) ◽  
pp. 10106
Author(s):  
Sanne Mol ◽  
Florianne M. J. Hafkamp ◽  
Laura Varela ◽  
Neena Simkhada ◽  
Esther W. Taanman-Kueter ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Peripheral Blood ◽  
Extracellular Vesicle ◽  
Neutrophil Activation ◽  
Imaging Method ◽  
Ros Production ◽  
Step Process ◽  
Gm Csf ◽  
Imaging Flow Cytometry ◽  
Blood Neutrophils

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli.

scholarly journals Monitoring Extracellular Vesicle Cargo Active Uptake by Imaging Flow Cytometry

2018 ◽  
Vol 9 ◽  
Author(s):  
Yifat Ofir-Birin ◽  
Paula Abou karam ◽  
Ariel Rudik ◽  
Tal Giladi ◽  
Ziv Porat ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicle ◽  
Active Uptake ◽  
Imaging Flow Cytometry

scholarly journals Fluorescence-Based Nanoparticle Tracking Analysis and Flow Cytometry for Characterization of Endothelial Extracellular Vesicle Release

2020 ◽  
Vol 21 (23) ◽  
pp. 9278
Author(s):  
Johannes Oesterreicher ◽  
Marianne Pultar ◽  
Jaana Schneider ◽  
Severin Mühleder ◽  
Johannes Zipperle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Cell Density ◽  
Current Knowledge ◽  
Umbilical Vein ◽  
Extracellular Vesicle ◽  
Nanoparticle Tracking Analysis ◽  
Specific Detection ◽  
Umbilical Vein Endothelial Cells

As extracellular vesicles (EVs) have become a prominent topic in life sciences, a growing number of studies are published on a regular basis addressing their biological relevance and possible applications. Nevertheless, the fundamental question of the true vesicular nature as well as possible influences on the EV secretion behavior have often been not adequately addressed. Furthermore, research regarding endothelial cell-derived EVs (EndoEVs) often focused on the large vesicular fractions comprising of microvesicles (MV) and apoptotic bodies. In this study we aimed to further extend the current knowledge of the influence of pre-isolation conditions, such as cell density and conditioning time, on EndoEV release from human umbilical vein endothelial cells (HUVECs). We combined fluorescence nanoparticle tracking analysis (NTA) and the established fluorescence-triggered flow cytometry (FT-FC) protocol to allow vesicle-specific detection and characterization of size and surface markers. We found significant effects of cell density and conditioning time on both abundance and size distribution of EndoEVs. Additionally, we present detailed information regarding the surface marker display on EVs from different fractions and size ranges. Our data provide crucial relevance for future projects aiming to elucidate EV secretion behavior of endothelial cells. Moreover, we show that the influence of different conditioning parameters on the nature of EndoEVs has to be considered.

scholarly journals Increased expression of six-large extracellular vesicle-derived miRNAs signature for nonvalvular atrial fibrillation

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Panjaree Siwaponanan ◽  
Pontawee Kaewkumdee ◽  
Wilasinee Phromawan ◽  
Suthipol Udompunturak ◽  
Nusara Chomanee ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Flow Cytometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Activation ◽  
Target Genes ◽  
Digital Pcr ◽  
Extracellular Vesicle ◽  
Nonvalvular Atrial Fibrillation

Abstract Backgrounds Non-valvular atrial fibrillation (AF) is the most common type of cardiac arrhythmia. AF is caused by electrophysiological abnormalities and alteration of atrial tissues, which leads to the generation of abnormal electrical impulses. Extracellular vesicles (EVs) are membrane-bound vesicles released by all cell types. Large EVs (lEVs) are secreted by the outward budding of the plasma membrane during cell activation or cell stress. lEVs are thought to act as vehicles for miRNAs to modulate cardiovascular function, and to be involved in the pathophysiology of cardiovascular diseases (CVDs), including AF. This study identified lEV-miRNAs that were differentially expressed between AF patients and non-AF controls. Methods lEVs were isolated by differential centrifugation and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), flow cytometry and Western blot analysis. For the discovery phase, 12 AF patients and 12 non-AF controls were enrolled to determine lEV-miRNA profile using quantitative reverse transcription polymerase chain reaction array. The candidate miRNAs were confirmed their expression in a validation cohort using droplet digital PCR (30 AF, 30 controls). Bioinformatics analysis was used to predict their target genes and functional pathways. Results TEM, NTA and flow cytometry demonstrated that lEVs presented as cup shape vesicles with a size ranging from 100 to 1000 nm. AF patients had significantly higher levels of lEVs at the size of 101–200 nm than non-AF controls. Western blot analysis was used to confirm EV markers and showed the high level of cardiomyocyte expression (Caveolin-3) in lEVs from AF patients. Nineteen miRNAs were significantly higher (> twofold, p < 0.05) in AF patients compared to non-AF controls. Six highly expressed miRNAs (miR-106b-3p, miR-590-5p, miR-339-3p, miR-378-3p, miR-328-3p, and miR-532-3p) were selected to confirm their expression. Logistic regression analysis showed that increases in the levels of these 6 highly expressed miRNAs associated with AF. The possible functional roles of these lEV-miRNAs may involve in arrhythmogenesis, cell apoptosis, cell proliferation, oxygen hemostasis, and structural remodeling in AF. Conclusion Increased expression of six lEV-miRNAs reflects the pathophysiology of AF that may provide fundamental knowledge to develop the novel biomarkers for diagnosis or monitoring the patients with the high risk of AF.

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