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High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were â¼10 PE/â¼80 nm EV diameter for CytoFLEX and â¼10 PEs/â¼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.
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Vesículas Extracelulares , Citometria de Fluxo , Citometria de Fluxo/métodos , Citometria de Fluxo/instrumentação , Humanos , Vesículas Extracelulares/metabolismo , Neoplasias Colorretais/diagnóstico , Linhagem Celular Tumoral , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/instrumentaçãoRESUMO
Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation-based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography-based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography- and ultracentrifugation-based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications.
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Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation.
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MOTIVATION: Extracellular particles (EPs) are the focus of a rapidly growing area of exploration due to the widespread interest in understanding their roles in health and disease. However, despite the general need for EP data sharing and established community standards for data reporting, no standard repository for EP flow cytometry data captures rigor and minimum reporting standards such as those defined by MIFlowCyt-EV (https://doi.org/10.1080/20013078.2020.1713526). We sought to address this unmet need by developing the NanoFlow Repository. RESULTS: We have developed The NanoFlow Repository to provide the first implementation of the MIFlowCyt-EV framework. AVAILABILITY AND IMPLEMENTATION: The NanoFlow Repository is freely available and accessible online at https://genboree.org/nano-ui/. Public datasets can be explored and downloaded at https://genboree.org/nano-ui/ld/datasets. The NanoFlow Repository's backend is built using the Genboree software stack that powers the ClinGen Resource, specifically the Linked Data Hub (LDH), a REST API framework written in Node.js, developed initially to aggregate data within ClinGen (https://ldh.clinicalgenome.org/ldh/ui/about). NanoFlow's LDH (NanoAPI) is available at https://genboree.org/nano-api/srvc. NanoAPI is supported by a Node.js Genboree authentication and authorization service (GbAuth), a graph database called ArangoDB, and an Apache Pulsar message queue (NanoMQ) to manage data inflows into NanoAPI. The website for NanoFlow Repository is built with Vue.js and Node.js (NanoUI) and supports all major browsers.
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Software , Bases de Dados Factuais , Citometria de FluxoRESUMO
Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.
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Eritrócitos/metabolismo , Vesículas Extracelulares/metabolismo , Insuficiência Cardíaca/sangue , Infarto do Miocárdio/sangue , Miocárdio/metabolismo , RNA Nuclear/genética , RNA-Seq/métodos , Transdução de Sinais/genética , Análise de Célula Única/métodos , Animais , Comunicação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/metabolismoRESUMO
MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences.
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Técnicas Biossensoriais , MicroRNAs , Regulação da Expressão Gênica , MicroRNAs/genética , Hibridização de Ácido NucleicoRESUMO
The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.
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Antígenos/análise , Células Sanguíneas , Magnetismo , Smartphone , Células Sanguíneas/química , Células Sanguíneas/citologia , Células Sanguíneas/imunologia , Contagem de Células , Humanos , Fenômenos MagnéticosRESUMO
RATIONALE: Previous translational studies implicate plasma extracellular microRNA-30d (miR-30d) as a biomarker in left ventricular remodeling and clinical outcome in heart failure (HF) patients, although precise mechanisms remain obscure. OBJECTIVE: To investigate the mechanism of miR-30d-mediated cardioprotection in HF. METHODS AND RESULTS: In rat and mouse models of ischemic HF, we show that miR-30d gain of function (genetic, lentivirus, or agomiR-mediated) improves cardiac function, decreases myocardial fibrosis, and attenuates cardiomyocyte (CM) apoptosis. Genetic or locked nucleic acid-based knock-down of miR-30d expression potentiates pathological left ventricular remodeling, with increased dysfunction, fibrosis, and cardiomyocyte death. RNA sequencing of in vitro miR-30d gain and loss of function, together with bioinformatic prediction and experimental validation in cardiac myocytes and fibroblasts, were used to identify and validate direct targets of miR-30d. miR-30d expression is selectively enriched in cardiomyocytes, induced by hypoxic stress and is acutely protective, targeting MAP4K4 (mitogen-associate protein kinase 4) to ameliorate apoptosis. Moreover, miR-30d is secreted primarily in extracellular vesicles by cardiomyocytes and inhibits fibroblast proliferation and activation by directly targeting integrin α5 in the acute phase via paracrine signaling to cardiac fibroblasts. In the chronic phase of ischemic remodeling, lower expression of miR-30d in the heart and plasma extracellular vesicles is associated with adverse remodeling in rodent models and human subjects and is linked to whole-blood expression of genes implicated in fibrosis and inflammation, consistent with observations in model systems. CONCLUSIONS: These findings provide the mechanistic underpinning for the cardioprotective association of miR-30d in human HF. More broadly, our findings support an emerging paradigm involving intercellular communication of extracellular vesicle-contained miRNAs (microRNAs) to transregulate distinct signaling pathways across cell types. Functionally validated RNA biomarkers and their signaling networks may warrant further investigation as novel therapeutic targets in HF.
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MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Comunicação Parácrina , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais , Quinase Induzida por NF-kappaBRESUMO
Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets.
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The original version of this article unfortunately contained a mistake.
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BACKGROUND: Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed. METHODS: We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS. FINDINGS: We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (nâ¯=â¯2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (pâ¯=â¯0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (nâ¯=â¯4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (pâ¯=â¯0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2â¯â¯=â¯0.888). INTERPRETATION: Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.
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Vesículas Extracelulares/metabolismo , Corantes Fluorescentes/administração & dosagem , Glioma/metabolismo , Ácidos Levulínicos/administração & dosagem , Fármacos Fotossensibilizantes/metabolismo , Protoporfirinas/metabolismo , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Glioma/diagnóstico , Glioma/cirurgia , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Imagem Óptica/métodos , Cirurgia Assistida por Computador , Ácido AminolevulínicoRESUMO
Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the "reference noise"). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.
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BACKGROUND/PURPOSE: Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases. METHODS: Exosomes were isolated by reagent-enhanced centrifugation from cell culture media of banked human bone marrow (bm) and amniotic fluid (af) MSCs after serum starvation. Characterization was by flow exometry for tetraspanin markers CD9, CD63, and CD81, transmission electron microscopy for size and morphology, and tunable resistive pulse sensing for size distribution and concentration. Statistical comparisons of count data were made by Poisson regression modeling and Student's T-test. RESULTS: Exosomes of appropriate size and morphology were isolated with comparable expressions of CD9 (96% vs. 94%), CD63 (88% vs. 66%), and CD81 (71% vs. 63%) for bmMSC and afMSC, respectively. Total exosome yield (particles/mL) adjusted for number of cells was higher from afMSCs than bmMSCs by an estimated 25% (Pâ¯<â¯0.001). CONCLUSIONS: While bone marrow and amniotic fluid mesenchymal stem cells are comparable sources of exosomes in size distribution, morphology, and expression of typical surface markers, yield may be higher from amniotic fluid cells. The amniotic fluid appears to be a preferable source of exosomes for clinical applications. LEVEL OF EVIDENCE: N/A (bench laboratory study).
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Líquido Amniótico/citologia , Medula Óssea/metabolismo , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Técnicas de Cultura de Células , Citometria de Fluxo/métodos , HumanosRESUMO
Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.
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Separação Celular/métodos , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Células JurkatRESUMO
During their lifetime, like all other cell types, red blood cells (RBCs) release both exosomes and plasma membrane derived EVs (ectosomes). RBC exosomes are formed only during the development of RBCs in bone marrow, and are released following the fusion of microvesicular bodies (MVB) with the plasma membrane. On the other hand, RBC EVs are generated during normal aging of RBCs in circulation by budding of the plasma membrane due to complement -mediated calcium influx, followed by vesicle shedding. This makes red blood cells and stored red cells a reliable source of EVs for basic and clinical research.
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Eritrócitos/metabolismo , Vesículas Extracelulares/metabolismo , Animais , Transporte Biológico , Preservação de Sangue , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Eritrócitos/imunologia , Eritrócitos/ultraestrutura , Exossomos/metabolismo , Vesículas Extracelulares/ultraestrutura , HumanosRESUMO
Here, we describe a comprehensive methodology for the setup and standardization of EV analysis using nanoscale flow cytometry. Controls of different size ranges, fluorescent intensities, and materials can be used to set up distribution curves that are then used for instrument optimization and as a reference guide. Using these controls, flow cytometry instruments can be primed for the detection, analysis, and sorting of specific EV populations. This allows for cross platform comparison and the ability to monitor both quality control (QC) and quality assurance (QA). The method here describes the use of nanoparticles to optimize a flow cytometer for small particle detection. It also outlines the procedures necessary to recover EVs for downstream applications.
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Vesículas Extracelulares/metabolismo , Citometria de Fluxo , Citometria de Fluxo/métodos , Microscopia de Fluorescência , Nanopartículas/química , Poliestirenos/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise EspectralRESUMO
Extracellular vesicles (EVs) serve an important function as mediators of intercellular communication. Exercise is protective for the heart, although the signaling mechanisms that mediate this cardioprotection have not been fully elucidated. Here using nano-flow cytometry, we found a rapid increase in plasma EVs in human subjects undergoing exercise stress testing. We subsequently identified that serum EVs were increased by ~1.85-fold in mice after 3-week swimming. Intramyocardial injection of equivalent quantities of EVs from exercised mice and non-exercised controls provided similar protective effects against acute ischemia/reperfusion (I/R) injury in mice. However, injection of exercise-induced EVs in a quantity equivalent to the increase seen with exercise (1.85 swim group) significantly enhanced the protective effect. Similarly, treatment with exercise-induced increased EVs provided additional anti-apoptotic effect in H2O2-treated H9C2 cardiomyocytes mediated by the activation of ERK1/2 and HSP27 signaling. Finally, by treating H9C2 cells with insulin-like growth factor-1 to mimic exercise stimulus in vitro, we found an increased release of EVs from cardiomyocytes associated with ALIX and RAB35 activation. Collectively, our results show that exercise-induced increase in circulating EVs enhances the protective effects of endogenous EVs against cardiac I/R injury. Exercise-derived EVs might serve as a potent therapy for myocardial injury in the future.
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Vesículas Extracelulares/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Condicionamento Físico Animal/métodos , Esforço Físico , Animais , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Teste de Esforço , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vesículas Extracelulares/transplante , Citometria de Fluxo/métodos , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/sangue , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Nanotecnologia/métodos , Estresse Oxidativo , Ratos , Natação , Fatores de Tempo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking, and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs), very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1) and tumor necrosis factor alpha (TNF-α). EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM) and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVs) outwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells. TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20 to 1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune responses. The contribution of the eosinophil-derived MVs to the regulation of immune responses awaits further investigation.
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The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.