Analysis of extracellular vesicles by flow cytometry – basics, limitations and prospects

2019 ◽  
Vol 1 (1) ◽  
pp. 40-45
Author(s):  
Andreas Spittler ◽  
Andre Görgens
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Size Range ◽  
Biological Fluids ◽  
Surface Protein ◽  
Cell Culture Supernatant ◽  
Protein Markers ◽  
Flow Cytometry Data ◽  
Surface Marker Expression ◽  
Established Technique

Flow cytometry is a well-established technique that classically is used to detect cells and quantify related parameters on the cellular surface, e. g. the expression of surface protein markers. Within the last few years, there also have been considerable advances of using flow cytometry to detect extracellular vesicles (EVs) in the size-range of exosomes and quantify their concentration and surface marker expression in EV-containing samples such as cell culture supernatant or biological fluids. Of note, such analyses of EVs within a size range well beyond <300 nm are still challenging and require a suitable instrumentation and a certain level of expertise to make sure essential controls are included and to ensure that resulting datasets can be interpreted appropriately. After giving a brief, basic introduction into flow cytometry which is essential especially for non-experienced readers in order to understand submicron particle flow cytometry data, we will give a concise overview about current possibilities and methods available in the field.

Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

scholarly journals Proteomic analysis of extracellular vesicles in cerebrospinal fluid of patients with Alzheimer’s disease

2020 ◽  
Author(s):  
Davide Chiasserini ◽  
Irene Bijnsdorp ◽  
Giovanni Bellomo ◽  
Pier Luigi Orvietani ◽  
Sander R. Piersma ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cerebrospinal Fluid ◽  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Neurodegenerative Disorder ◽  
High Resolution Mass Spectrometry ◽  
Biological Fluids ◽  
Protein Profile ◽  
Protein Markers

AbstractCerebrospinal fluid (CSF) contains different types of extracellular vesicles (EVs) with undisclosed biomarker potential for neurodegenerative diseases. The aims of the present study were: i) to compare the proteome EVs isolated using different ultracentrifugation speed ii) to preliminary explore the EVs proteome in a common neurodegenerative disorder, Alzheimer’s disease (AD) compared to neurological controls. CSF samples from control subjects and AD patients were pooled separately (15 mL) and subjected to ultracentrifugation (UC) at different speeds (20,000g and 100,000g) to isolate separate EV fractions (P20 and P100). The proteome was analysed using high-resolution mass spectrometry (LC-MS/MS) and comparisons were made using bioinformatic analysis. EVs isolated at 100,000g (P100) had a proteome consistent with vesicles secreted via an ESCRT-dependent mechanism, being highly enriched in alix (PDCD6IP), syntenin-1 (SDCBP) and TSG101. EVs isolated at 20,000g were substantially different, showing enrichment in cytoskeletal and cell adhesion molecules. The pools from patients diagnosed with AD showed a distinct protein profile of CSF EVs, with increased levels of ADAM10, SPON1, CH3IL1 and MDK in the P100 fraction. CSF EV offer a new potential biosource of protein markers for AD detection and a complementary framework to the analysis of whole biological fluids for biomarker discovery.

An Emerging Fluorescence-Based Technique for Quantification and Protein Profiling of Extracellular Vesicles

2020 ◽  
pp. 247263032097045
Author(s):  
Mehdi Dehghani ◽  
Rebecca K. Montange ◽  
Michael W. Olszowy ◽  
David Pollard
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Protein Profiling ◽  
Submicron Particles ◽  
Protein Markers ◽  
Bulk Analysis ◽  
Precise Quantification ◽  
Therapeutic Tools ◽  
Different Sources

Robust and well-established techniques for the quantification and characterization of extracellular vesicles (EVs) are a crucial need for the utilization of EVs as potential diagnostic and therapeutic tools. Current bulk analysis techniques such as proteomics and Western blot suffer from low resolution in the detection of small changes in target marker expression levels, exemplified by the heterogeneity of EVs. Microscopy-based techniques can provide valuable information from individual EVs; however, they are time-consuming and statistically less powerful than other techniques. Flow cytometry has been successfully employed for the quantification and characterization of individual EVs within larger populations. However, traditional flow cytometry is not highly suited for the examination of smaller, submicron particles. Here we demonstrate the accurate and precise quantification of nanoparticles such as EVs using the Virus Counter 3100 (VC3100) platform, a fluorescence-based technique that uses the principles of flow cytometry with critical enhancements to enable the effective detection of smaller particles. This approach can detect nanoparticles precisely with no evidence of inaccurate concentration measurement from masking effects associated with traditional nanoparticle tracking analysis (NTA). Fluorescently labeled EVs from different sources were successfully quantified using the VC3100 without a postlabeling washing step. Moreover, protein profiling and characterization of individual EVs were achieved and have been shown to determine the expression level of target protein markers.

scholarly journals Extracellular Vesicles: Current Analytical Techniques for Detection and Quantification

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 824 ◽  
Author(s):  
Esther Serrano-Pertierra ◽  
Myriam Oliveira-Rodríguez ◽  
María Matos ◽  
Gemma Gutiérrez ◽  
Amanda Moyano ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Supernatant ◽  
Biological Fluids ◽  
Analytical Techniques ◽  
Response To Treatment ◽  
Cell Culture Supernatant ◽  
Advantages And Disadvantages ◽  
Potential Applications ◽  
Detection And Quantification ◽  
Potential Biomarkers

Since their first observation, understanding the biology of extracellular vesicles (EV) has been an important and challenging field of study. They play a key role in the intercellular communication and are involved in important physiological and pathological functions. Therefore, EV are considered as potential biomarkers for diagnosis, prognosis, and monitoring the response to treatment in some diseases. In addition, due to their properties, EV may be used for therapeutic purposes. In the study of EV, three major points have to be addressed: 1. How to isolate EV from cell culture supernatant/biological fluids, 2. how to detect them, and 3. how to characterize and quantify. In this review, we focus on the last two questions and provide the main analytical techniques up-to-date for detection and profiling of EV. We critically analyze the advantages and disadvantages of each one, aimed to be of relevance for all researchers working on EV biology and their potential applications.

scholarly journals Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

scholarly journals Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis

2021 ◽  
Vol 22 (8) ◽  
pp. 3839
Author(s):  
Sobha Karuthedom Karuthedom George ◽  
Lucia Lauková ◽  
René Weiss ◽  
Vladislav Semak ◽  
Birgit Fendl ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Comparative Analysis ◽  
Extracellular Vesicles ◽  
High Throughput ◽  
Single Particle ◽  
Size Range ◽  
Detection Limits ◽  
Nanoparticle Tracking Analysis ◽  
Fluorescent Beads ◽  
Good Agreement

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

scholarly journals Quantitative Proteomic Profiling of Small Molecule Treated Mesenchymal Stem Cells Using Chemical Probes

2020 ◽  
Vol 22 (1) ◽  
pp. 160
Author(s):  
Jerran Santos ◽  
Sibasish Dolai ◽  
Matthew B. O’Rourke ◽  
Fei Liu ◽  
Matthew P. Padula ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ribosomal Proteins ◽  
Surface Protein ◽  
Temporal Analysis ◽  
Phenotypic Characterization ◽  
Major Protein ◽  
Adipose Derived Stem Cells ◽  
Protein Markers ◽  
Proteomic Profiling ◽  
Depth Analysis

The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation.

scholarly journals Conventional, High-Resolution and Imaging Flow Cytometry: Benchmarking Performance in Characterisation of Extracellular Vesicles

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 124
Author(s):  
Jaco Botha ◽  
Haley R. Pugsley ◽  
Aase Handberg
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Extracellular Vesicles ◽  
Single Particles ◽  
Multiple Parameters ◽  
Imaging Flow Cytometry ◽  
Highly Sensitive ◽  
Individual Project ◽  
Benchmarking Performance ◽  
High Throughput Manner

Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.

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