scholarly journals The Significance of Phenotyping and Quantification of Plasma Extracellular Vesicles Levels Using High-Sensitivity Flow Cytometry during COVID-19 Treatment

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 767
Author(s):  
Igor Kudryavtsev ◽  
Olga Kalinina ◽  
Vadim Bezrukikh ◽  
Olesya Melnik ◽  
Alexey Golovkin
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Dynamic Assessment ◽  
Severe Infection ◽  
High Sensitivity ◽  
Cell Types ◽  
Significant Loss ◽  
Therapy Effectiveness ◽  
Scientific Testing ◽  
Different Cell Types

New investigation results point to the potential participation of extracellular vesicles (EVs) in the pathogenesis of coronavirus infection, its progression, and mechanisms of the therapy effectiveness. This dictates the necessity to transfer scientific testing technologies to medical practice. Here, we demonstrated the method of phenotyping and quantitative analysis of plasma EVs based on differential centrifugation, immunostaining, and high-sensitivity multicolor flow cytometry. We used EV markers that were potentially associated with SARS-CoV-2 dissemination via vesicles and cell-origination markers, characterizing objects from different cell types that could influence clinical manifestation of COVID-19. Plasma levels of CD235a+ and CD14+ EVs in patients with moderate infection were significantly increased while CD8+ and CD19+ EVs were decreased comparing with HD. Patients with severe infection had lower levels of CD4+, CD19+, and CD146+ EVs than HD. These findings demonstrate that EV concentrations in COVID-19 are severity related. Moreover, the three-point dynamic assessment demonstrated significant loss of CD63+ and CD147+ plasma EVs. The used method can be a convenient tool for vital infection pathogenesis investigation and for COVID-19 diagnostics.

Extracellular vesicles in the circulation: are erythrocyte microvesicles a confounder in the plasma haemoglobin assay?

2013 ◽  
Vol 41 (1) ◽  
pp. 288-292 ◽  
Author(s):  
Karen M.K. de Vooght ◽  
Cedric Lau ◽  
Pim P.M. de Laat ◽  
Richard van Wijk ◽  
Wouter W. van Solinge ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Formation Rate ◽  
Haemoglobin Concentration ◽  
Cell Types ◽  
Total Cell ◽  
Plasma Haemoglobin ◽  
Free Plasma Haemoglobin ◽  
Free Haemoglobin ◽  
Different Cell Types ◽  
Free Plasma

Blood contains a mixture of extracellular vesicles from different cell types, primarily platelets, endothelial cells, leucocytes and erythrocytes. Erythrocytes are the most abundant cell type in blood and could, especially in certain pathologies, represent an important source of vesicles. Since erythrocytes contain the haemoglobin components iron and haem, which are potentially toxic, it is important to investigate the contribution of vesicle-associated haemoglobin to total cell-free haemoglobin levels. To our knowledge, this is the first time that cell-free plasma haemoglobin has been differentiated into vesicle-associated and molecular species. We investigated the contribution of vesicle-associated haemoglobin in residual patient material that was routinely analysed for total cell-free plasma haemoglobin. All patient samples included in the study were haemolytic with total cell-free haemoglobin concentration ranging from 80 to 2500 mg/l. In the majority of the samples, total cell-free haemoglobin concentration was between 100 and 200 mg/l. No haemoglobin could be detected in the vesicle fraction, indicating that the contribution of vesicle-associated haemoglobin to total cell free-haemoglobin levels in plasma is negligible. It is important to investigate whether erythrocyte vesicles are not formed in blood or that their production is not increased during pathologies associated with haemolysis or that the clearance rate of the vesicles surpasses the formation rate.

scholarly journals Contributions of Flow Cytometry to the Molecular Study of Spermatogenesis in Mammals

2021 ◽  
Vol 22 (3) ◽  
pp. 1151 ◽  
Author(s):  
Rosana Rodríguez-Casuriaga ◽  
Adriana Geisinger
Keyword(s):  
Flow Cytometry ◽  
Basic Research ◽  
Cell Types ◽  
Molecular Study ◽  
Culture Systems ◽  
Invaluable Tool ◽  
Mutant Phenotypes ◽  
Different Cell Types

Mammalian testes are very heterogeneous organs, with a high number of different cell types. Testicular heterogeneity, together with the lack of reliable in vitro culture systems of spermatogenic cells, have been an obstacle for the characterization of the molecular bases of the unique events that take place along the different spermatogenic stages. In this context, flow cytometry has become an invaluable tool for the analysis of testicular heterogeneity, and for the purification of stage-specific spermatogenic cell populations, both for basic research and for clinical applications. In this review, we highlight the importance of flow cytometry for the advances on the knowledge of the molecular groundwork of spermatogenesis in mammals. Moreover, we provide examples of different approaches to the study of spermatogenesis that have benefited from flow cytometry, including the characterization of mutant phenotypes, transcriptomics, epigenetic and genome-wide chromatin studies, and the attempts to establish cell culture systems for research and/or clinical aims such as infertility treatment.

scholarly journals Extracellular Vesicles in Bone Tumors: How to Seed in the Surroundings Molecular Information for Malignant Transformation and Progression

2021 ◽  
Vol 11 ◽  
Author(s):  
Alfredo Cappariello ◽  
Nadia Rucci
Keyword(s):  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Cell Types ◽  
Biologically Active ◽  
Main Body ◽  
Bone Microenvironment ◽  
Premetastatic Niche ◽  
Favourable Environment ◽  
Cell Dormancy ◽  
Different Cell Types

Bone is a very dynamic tissue hosting different cell types whose functions are regulated by a plethora of membrane-bound and soluble molecules. Intercellular communication was recently demonstrated to be also sustained by the exchange of extracellular vesicles (EVs). These are cell-derived nanosized structures shuttling biologically active molecules, such as nucleic acids and proteins. The bone microenvironment is a preferential site of primary and metastatic tumors, in which cancer cells find a fertile soil to “seed and blossom”. Nowadays, many oncogenic processes are recognized to be sustained by EVs. For example, EVs can directly fuel the vicious cycle in the bone/bone marrow microenvironment. EVs create a favourable environment for tumor growth by affecting osteoblasts, osteoclasts, osteocytes, adipocytes, leukocytes, and endothelial cells. At the same time other crucial tumor-mediated events, such as the premetastatic niche formation, tumor cell dormancy, as well as drug resistance, have been described to be fostered by tumor-derived EVs. In this review, we will discuss the main body of literature describing how the cancer cells use the EVs for their growth into the bone and for educating the bone microenvironment to host metastases.

scholarly journals Novel PE and APC Tandems: Additional Near-Infrared Fluorochromes for Use in Spectral Flow Cytometry

2020 ◽  
Author(s):  
Yekyung Seong ◽  
Tuan Andrew Nguyen ◽  
Yian Wu ◽  
Denny Nguyen ◽  
Archana Thakur ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Near Infrared ◽  
Biomarker Discovery ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Cell Types ◽  
Color Flow ◽  
Emission Signal ◽  
Avalanche Diodes

ABSTRACTFlow cytometry is an essential technology to identify and quantify cell populations. To support the need for high content analyses, we developed new fluorochromes designed specifically to enhance our flow cytometry capabilities. A standard Cytek Aurora flow cytometer has 3 excitation lasers (405nm, 488nm, and 640nm) and incorporates the latest Avalanche Diodes Photodetector (ADP) technology, which demonstrates significant improvement in sensitivity for fluorescent emission signals longer than 800nm. However, there are no commercially available fluorochromes capable of being excited by the above lasers with peak emission signal beyond 800nm. To address this gap, we engineered 6 new fluorochromes: PE-750, PE-800, PE-830 for the 488nm laser and APC-750, APC-800, APC-830 for the 640nm laser. These novel fluorescent molecules were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Via the principle of fluorescence resonance energy transfer (FRET), these conjugates create unique fluorochromes with the excitation properties of the donor and the emission properties of the acceptor. After the fluorochromes were created and validated, they were conjugated to a test antibody. We demonstrated long-term stability of the conjugated antibodies at −20°C with protein stabilizing cocktails. Most importantly, in order to show the utility of these novel fluorochromes, we created and validated a 34-color flow cytometry panel designed to measure broad human immune function with high sensitivity. This panel will be applied for immunophenotyping of multiple different cell types from dissociated tumors and blood samples. In conclusion, this novel 34-color high-content flow cytometry panel will be able to support enhanced analysis and diagnosis in Immuno-oncology and facilitate innovation in biomarker discovery.

scholarly journals Raman Fingerprint of Extracellular Vesicles and Conditioned Media for the Reproducibility Assessment of Cell-Free Therapeutics

2021 ◽  
Vol 9 ◽  
Author(s):  
Cristiano Carlomagno ◽  
Chiara Giannasi ◽  
Stefania Niada ◽  
Marzia Bedoni ◽  
Alice Gualerzi ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stromal Cells ◽  
Cell Types ◽  
Dermal Fibroblasts ◽  
Cell Secretion ◽  
Free Products ◽  
Regenerative Effect ◽  
Different Cell Types ◽  
Non Destructive

Extracellular Vesicles (EVs) and Conditioned Medium (CM) are promising cell-free approaches to repair damaged and diseased tissues for regenerative rehabilitation purposes. They both entail several advantages, mostly in terms of safety and handling, compared to the cell-based treatment. Despite the growing interest in both EVs and CM preparations, in the light of a clinical translation, a number of aspects still need to be addressed mainly because of limits in the reproducibility and reliability of the proposed protocols. Raman spectroscopy (RS) is a non-destructive vibrational investigation method that provides detailed information about the biochemical composition of a sample, with reported ability in bulk characterization of clusters of EVs from different cell types. In the present brief report, we acquired and compared the Raman spectra of the two most promising cell-free therapeutics, i.e., EVs and CM, derived from two cytotypes with a history in the field of regenerative medicine, adipose-derived mesenchymal stem/stromal cells (ASCs) and dermal fibroblasts (DFs). Our results show how RS can verify the reproducibility not only of EV isolation, but also of the whole CM, thus accounting for both the soluble and the vesicular components of cell secretion. RS can provide hints for the identification of the soluble factors that synergistically cooperate with EVs in the regenerative effect of CM. Still, we believe that the application of RS in the pipeline of cell-free products preparation for therapeutic purposes could help in accelerating translation to clinics and regulatory approval.

scholarly journals High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carmen Campos-Silva ◽  
Henar Suárez ◽  
Ricardo Jara-Acevedo ◽  
Estefanía Linares-Espinós ◽  
Luis Martinez-Piñeiro ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
High Sensitivity ◽  
High Sensitivity Detection ◽  
Sensitivity Detection

scholarly journals Apoptotic Bodies: Particular Extracellular Vesicles Involved in Intercellular Communication

Biology ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 21 ◽  
Author(s):  
Michela Battistelli ◽  
Elisabetta Falcieri
Keyword(s):  
Extracellular Vesicles ◽  
Intercellular Communication ◽  
Genetic Information ◽  
Cell Communication ◽  
Cell Types ◽  
Apoptotic Bodies ◽  
Species Barrier ◽  
Physiological Conditions ◽  
Different Types ◽  
Different Cell Types

In the last decade, a new method of cell–cell communication mediated by membranous extracellular vesicles (EVs) has emerged. EVs, including exosomes, microvesicles, and apoptotic bodies (ApoBDs), represent a new and important topic, because they are a means of communication between cells and they can also be involved in removing cellular contents. EVs are characterized by differences in size, origin, and content and different types have different functions. They appear as membranous sacs released by a variety of cells, in different physiological and patho-physiological conditions. Intringuingly, exosomes and microvesicles are a potent source of genetic information carriers between different cell types both within a species and even across a species barrier. New, and therefore still relatively poorly known vesicles are apoptotic bodies, on which numerous in-depth studies are needed in order to understand their role and possible function. In this review we would like to analyze their morpho-functional characteristics.

scholarly journals GLI ESOSOMI NELLA COMUNICAZIONE CELLULA-CELLULA

2020 ◽  
Author(s):  
Stefania Raimondo
Keyword(s):  
Small Molecules ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Cell Types ◽  
Cell Contact ◽  
Bioactive Lipids ◽  
Pathological Conditions ◽  
Extracellular Matrix Components ◽  
Different Cell Types ◽  
Cell Cell

Cell to cell communication is essential for the coordination and proper organization of different cell types in multicellular systems. Cells exchange information through a multitude of mechanisms such as secreted growth factors and chemokines, small molecules (peptides, ions, bioactive lipids and nucleotides), cell-cell contact and the secretion of extracellular matrix components. Over the last few years a new and sophisticated mechanism of cell-cell communication based on extracellular vesicles has been described. Extracellular vesicles are specialized vesicles released in the extracellular space by most of cell types, under physiological and pathological conditions. Among different extracellular vesicles subtypes, exosomes (30-100 nm) have recently received most of the attention do to their ability to be messenger in intercellular communication.

scholarly journals Extracellular Vesicles in Human Oogenesis and Implantation

2019 ◽  
Vol 20 (9) ◽  
pp. 2162 ◽  
Author(s):  
Francesca Andronico ◽  
Rosalia Battaglia ◽  
Marco Ragusa ◽  
Davide Barbagallo ◽  
Michele Purrello ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cell Communication ◽  
Cell Types ◽  
Female Reproduction ◽  
Reproductive Process ◽  
Complex Processes ◽  
Egg Maturation ◽  
Different Cell Types ◽  
Human Oogenesis

Reproduction, the ability to generate offspring, represents one of the most important biological processes, being essential for the conservation of the species. In mammals, it involves different cell types, tissues and organs, which, by several signaling molecules, coordinate the different events such as gametogenesis, fertilization and embryo development. In the last few years, the role of Extracellular Vesicles, as mediators of cell communication, has been investigated in every phase of these complex processes. Microvesicles and exosomes, identified in the fluid of ovarian follicles during egg maturation, are involved in communication between the developing oocyte and the somatic follicular cells. More recently, it has been demonstrated that, during implantation, Extracellular Vesicles could participate in the complex dialog between the embryo and maternal tissues. In this review, we will focus our attention on extracellular vesicles and their cargo in human female reproduction, mainly underlining the involvement of microRNAs in intercellular communication during the several phases of the reproductive process.

scholarly journals In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish

2021 ◽  
Author(s):  
Aaron Scott ◽  
Lorena Sueiro Ballesteros ◽  
Marston Bradshaw ◽  
Chisato Tsuji ◽  
Ann Power ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cell Types ◽  
Adult Zebrafish ◽  
Cell Type ◽  
Zebrafish Model ◽  
Cell Type Specific ◽  
In Vivo Characterization ◽  
Different Cell Types

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

Sign in / Sign up

Export Citation Format

Share Document