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.

Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry

ACS Nano ◽  
2018 ◽  
Vol 12 (1) ◽  
pp. 671-680 ◽  
Author(s):  
Ye Tian ◽  
Ling Ma ◽  
Manfei Gong ◽  
Guoqiang Su ◽  
Shaobin Zhu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Cancer Patients ◽  
Extracellular Vesicles ◽  
Protein Profiling ◽  
Colorectal Cancer Patients

scholarly journals Nanoscale flow cytometry of patient plasma for the detection of prostate cancer-associated extracellular vesicles

2017 ◽  
Vol 3 (2) ◽  
pp. 1-2 ◽  
Author(s):  
Andrew C. Poon ◽  
Johanna Garzon ◽  
Sabine Brett ◽  
Matthew Lowerison ◽  
Karla Williams ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
Benign Prostatic Hyperplasia ◽  
Cancer Patients ◽  
Extracellular Vesicles ◽  
Specific Antigen ◽  
Prostatic Hyperplasia ◽  
Submicron Particles ◽  
Screening Tests ◽  
Positive Events

Introduction Prostate cancer is the predominant cancer in men, affecting one in seven men during their lifetime. Current tests for prostate cancer include the digital rectal exam and the prostate-specific antigen (PSA) test. Extracellular vesicles (EVs) are submicron particles that participate in intercellular cross-talk by releasing cell mediators such as microRNA, carbohydrates and proteins. While they are known to express the broad tetraspanin family of proteins, i.e. CD9/CD63, prostate cancer-derived EVs have also been found to express PSA and six transmembrane epithelial antigen of the prostate (STEAP1). Traditionally, scientists have purified these EVs through ultracentrifugation. Here we propose a tandem purification of patient plasma, followed by nanoscale flow cytometry (A50+) as a novel method to detect tumour-derived EVs. Materials and Methods Plasma was obtained from healthy volunteers, patients with benign prostatic hyperplasia (BPH), and patients with metastatic prostate cancer. CD9, CD63, PSA and STEAP1 were used as primary antibodies for the purification of EVs from neat plasma. To perform the purification in tandem, Protein G immunoprecipitation using CD9 and PSA was carried out first, followed by immunoaffinity purification with biotinylated CD63 and STEAP1. First and second elutions were collected for the enumeration of dual positive events by A50+. Initial histogram overlays and bivariate plots of neat and purified plasma were computed, then exported as comma-separated values for mathematical modelling by MATLAB. Results Strong dual positive EV populations from patient plasma were optimised, demonstrating that the method enriches tumour-derived EVs from neat samples of patient plasma. This was observed for HVs, patients with benign prostatic hyperplasia, and prostate cancer patients with Gleason 4+4. Enrichment in these purified samples was measured by A50+ and demonstrated by overlaying purified and non-purified histoplots by MATLAB. Additional results show that PSA and STEAP1 can be adapted to detect single or dual positive populations of tumour-associated EVs in prostate cancer patients. Discussions and Conclusions This study suggests that tandem purification of tumour-associated EVs and A50+ analysis from plasma of prostate cancer patients can lead to earlier diagnosis and risk stratification, compared to traditional screening tests and aspiration cytology. Future studies will be directed toward optimizing this detection method for markers of other cancer types to achieve better outcomes in cancer detection and prognosis.

scholarly journals Platelet-Derived Extracellular Vesicles for Regenerative Medicine

2021 ◽  
Author(s):  
Miquel Antich-Rosselló ◽  
Maria Antònia Forteza-Genestra ◽  
Marta Monjo ◽  
Joana Maria Ramis
Keyword(s):  
Wound Healing ◽  
Regenerative Medicine ◽  
Extracellular Vesicles ◽  
Cellular Differentiation ◽  
Neural Regeneration ◽  
Isolation And Characterization ◽  
Biomedical Field ◽  
New Treatments ◽  
Different Sources

Extracellular vesicles (EVs) present a great potential for the development of new treatments in the biomedical field. To be used as therapeutics, many different sources have been used for EVs obtention, while only few studies have addressed the use of platelet derived EVs (pEVs). In fact, pEVs have been shown to intervene in different healing responses, thus some studies have evaluated their regenerative capability in wound healing or hemorrhagic shock. Even more, pEVs have proven to induce cellular differentiation, enhancing musculoskeletal or neural regeneration. However, the obtention and characterization of pEVs is widely heterogeneous and differs from the recommendations of the International Society for Extracellular Vesicles. Therefore, in this review, we aim to present the main advances in the therapeutical use of pEVs in the regenerative medicine field while highlighting the isolation and characterization steps followed. The main goal of this review is to portray the studies performed in order to enhance the translation of the pEVs research into feasible therapeutical applications.

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.

scholarly journals Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours

2019 ◽  
Vol 8 (1) ◽  
pp. 1588555 ◽  
Author(s):  
Franz L. Ricklefs ◽  
Cecile L. Maire ◽  
Rudolph Reimer ◽  
Lasse Dührsen ◽  
Katharina Kolbe ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Brain Tumours ◽  
Imaging Flow Cytometry ◽  
Malignant Brain Tumours ◽  
Multiparametric Characterization

scholarly journals Population Analysis of Extracellular Vesicles in Microvolumes of Biofluids

2020 ◽  
Author(s):  
Joana Maia ◽  
Silvia Batista ◽  
Nuno Couto ◽  
Ana C. Gregório ◽  
Cristian Bodo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Population Analysis ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Sample Volume ◽  
Clinical Samples ◽  
Liquid Biopsies ◽  
Clinical Biomarkers

AbstractExtracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. Although frequently employed for content characterization of EVs, the study of bulk preparations lacks information on sub-populations and the intrinsic heterogeneity of vesicles. Importantly, these strategies also difficult the characterization of EVs from small quantities of samples. We here present a Flow Cytometry strategy that enables detailed population analysis of EVs, at the same time decreasing sample volume requirements and accelerating the overall processing time. We show its unique application for quality control of isolates of EVs by comparing the proportion of vesicular and non-vesicular particles in samples prepared by different protocols. In addition, we demonstrate its suitability for the study of populations of EVs from samples characterized by challenging small volumes. To illustrate that, we perform longitudinal non-lethal analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By allowing for the analysis of EVs from minimal amounts of sample, our Flow Cytometry strategy has an unexplored potential in the study of EVs in clinical samples with intrinsically limited volumes. When compared to conventional methods, it also multiplies by several times the number of different analytes that can be studied from a single collection of biofluid.

scholarly journals Antibody-Free Labeling of Malaria-Derived Extracellular Vesicles Using Flow Cytometry

Biomedicines ◽  
2020 ◽  
Vol 8 (5) ◽  
pp. 98
Author(s):  
Elya Dekel ◽  
Paula Abou Karam ◽  
Yael Ohana-Daniel ◽  
Mirit Biton ◽  
Neta Regev-Rudzki ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Intercellular Communication ◽  
Malaria Parasite ◽  
Flow Cytometer ◽  
Cellular Processes ◽  
Parasite Plasmodium ◽  
Membrane Bound ◽  
The Way

Extracellular vesicles (EVs) are cell-derived membrane-bound structures that are believed to play a major role in intercellular communication by allowing cells to exchange proteins and genetic cargo between them. In particular, pathogens, such as the malaria parasite Plasmodium (P.) falciparum, utilize EVs to promote their growth and to alter their host’s response. Thus, better characterization of these secreted organelles will enhance our understanding of the cellular processes that govern EVs’ biology and pathological functions. Here we present a method that utilizes a high-end flow cytometer system to characterize small EVs, i.e., with a diameter less than 200 nm. Using this method, we could evaluate different parasite-derived EV populations according to their distinct cargo by using antibody-free labeling. It further allows to closely monitor a sub-population of vesicles carrying parasitic DNA cargo. This ability paves the way to conducting a more ‘educated’ analysis of the various EV cargo components.

scholarly journals Surface Marker Expression in Small and Medium/Large Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Naive or Apoptotic Condition Using Orthogonal Techniques

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2948
Author(s):  
Renata Skovronova ◽  
Cristina Grange ◽  
Veronica Dimuccio ◽  
Maria Chiara Deregibus ◽  
Giovanni Camussi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Super Resolution ◽  
Analytical Techniques ◽  
Surface Marker ◽  
Bulk Analysis ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Super Resolution Microscopy ◽  
Different Sources

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a promising resource for regenerative medicine. Small MSC-EVs represent the active EV fraction. A bulk analysis was applied to characterise MSC-EVs’ identity and purity, with the assessment of single EV morphology, size and integrity using electron microscopy. We applied different methods to quantitatively analyse the size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC-EVs obtained using sequential ultracentrifugation. Bone marrow, adipose tissue and umbilical cord MSC-EVs were compared in naive and apoptotic conditions. As detected by electron microscopy, the 100k EV size < 100 nm was confirmed by super-resolution microscopy and ExoView. Single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanins. ExoView allowed a comparative screening of single MSC-EV tetraspanin and mesenchymal markers. A semiquantitative bead-based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC-EVs. Apoptotic MSC-EVs were released in higher numbers, without significant differences in the naive fractions in surface marker expression. These results show a consistent profile of MSC-EV fractions among the different sources and a safer profile of the 100k MSC-EV population for clinical application. Our study identified suitable applications for EV analytical techniques.

Sign in / Sign up

Export Citation Format

Share Document