Abstract: Study Design An experimental study with extracellular vesicles (EVs) from mesenchymal stem cell (MSC) of the epidural fat (EF) of the spine. Purpose This study aims to isolate the exosomes from epidural fat-derived mesenchymal stem cells (EF-MSCs) and fully characterize the EF-MSC-EVs. Overview of Literature EF-MSCs were reported in 2019, and a few studies have shown the positive outcomes of using EF-MSCs to treat specific spine pathologies. However, MSCs have significant limitations for conducting basic studies or developing therapeutic agents. Although EVs are an emerging research topic, no studies have focused on EVs, especially exosomes, from EF and EF-MSCs. Methods In this study, we isolated the exosomes using the tangential flow filtration (TFF) system with exosome-depleted fetal bovine serum and performed the characterization tests via western blotting, reverse transcription-polymerase chain reaction, nanoparticle tracking analysis (NTA), and transmission electron microscopy. Results In transmission electron microscopy, the exosome had a diameter of approximately 100-200 nm and had a spherical shape, whereas in the NTA, the exosome had an average diameter of 142.8 nm with a concentration of 1.27×1010 particles/mL. The flow cytometry analysis showed the expression of CD63 and CD81. The western blotting analysis showed the positive markers. Conclusions These findings showed that isolating the exosomes via TFF resulted in high-quality EF-MSC exosome yield. Further studies with exosomes from EF-MSC are needed to evaluate the function and role of the EF tissue.

Abstract: The present invention relates to a diagnostic method where protein complexes are applied as biomarkers for autoimmune diseases, preferably SLE and arthritis. The method applies nanoparticle tracking analysis (NTA) preferably in combination antibody-conjugated fluorescent nanoparticles (e.g. "quantum dots") to detect large proteins complexes, such as complexes of mannose-binding lectin (MBL) and/or Gal-3 protein in human plasma.

Abstract: Exosomes are nanoscale (30–150 nm) biological vesicles that are actively released from living cells and circulating into all body fluids. Recently, exosomes in cerebrospinal fluid (CSF) have been recognized as promising biomarkers for central nervous system (CNS) neoplasms. In this study, we report a label-free method that can be used to rapidly isolate exosomes from CSF for proteomic analysis. Compared to ultracentrifugation and polyethylene glycol-based precipitation, our method isolates exosomes from 2 mL of CSF within 10 min, which is 18 times and 72 times shorter, respectively; the yield was increased by 4.47 times and 2.09 times and the purity was increased by 4.54 times and 9.76 times, respectively. The proteomic analysis further revealed that the exosomes isolated by our method identified more exosome-related proteins, which may reflect the physiological status of diseases for exosome-based diagnosis. Therefore, the effective isolation of pure exosomes from CSF samples for protein analysis will benefit the downstream analysis and clinical translation of exosomes, thus promoting the early diagnosis of CNS neoplasms.

Abstract: We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.

Abstract: Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 10$^{10}$ EVs/ml and 6.0 × 10$^{10}$ EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p < .05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development.

Abstract: Extracellular vesicles (EVs) are membrane-bound nanoparticles that are secreted by most cell types with an emerging role in cellular communication and potential as biomarkers of disease. Nanoparticle tracking analysis (NTA) is a commonly used technique to measure the size and concentration of nanoparticles, such as EVs. Here, we present two protocols for the analysis of size profile concentration, and zeta potential (ZP) of well-characterized EVs derived from human choriocarcinoma JAr cells using NTA. These protocols describe how the size profile concentration, and ZP of JAr EVs are measured using optimized settings of NTA. With good experimental practices and consistent protocol, NTA measurements of EVs can provide reliable data that could potentially translate further uses of EVs for diagnostic and therapeutic applications.

Abstract: Additional file 1: Fig. S1. Characterization of AuNPs, AuNPs-NH2. TEM images of AuNPs (a-1) and AuNPs-NH2 (a-2). Size distribution and size in water of AuNPs (b-1) and AuNPs-NH2 (b-2). c Zeta-potentials of AuNPs and AuNPs-NH2. d Spectrophotometer results of AuNPs and AuNPs-NH2. e In vitro toxicity evaluation of U87-MG cells after co-incubation with AuNPs-NH2 for 24 h by Cell Counting Kit-8 (CCK-8) method. f Dynamic light scattering (DLS) size measurements of AuNPs-NH2 in PBS for varied time durations (0 ~ 29 days). Fig. S2. Characterization of Au-DOX. IC50 of DOX (a) and AuNPs combined with radiotherapy of 6Gy (b). c TEM image of Au-DOX. d Spectrophotometer results of AuNPs , AuNPs-NH2 and Au-DOX. e FT-IR spectrum of Au-DOX. f Size distribution and size in water of Au-DOX. Fig. S3. Characterization of PCL-PEOz- maleimide. a XPS spectra of PCL-PEOz- maleimide. b 1HNMR spectrum of the polymer. Fig. S4. Photographs of blank polymersomes (a-1) and cargo-loaded polymersomes (a-2). TEM images of blank polymersomes (b-1) and cargo-loading polymersomes (b-2).c In vitro toxicity evaluation of U87-MG cells after co-incubation with PO and ANG-PO for 24 h by Cell Counting Kit-8 (CCK-8) method. Fig. S5. The particle number of polymersomes was measured using NTA. The original sample was diluted 250 times before testing. The particle number of original sample was yielded a value of 6×1011 particles/mL after calculation. Fig. S6. Identification of primary astrocytes (a-1) and cerebral microvascular endothelial cells (a-2) by flow cytometry. b Expression levels of LRP1 on cell lines of U87-MG, BMECs, and normal astrocyte determined by Western blot. c Quantitative analysis of LRP1 protein levels. Data are presented as the mean plus or minus the standard deviation (SD), and n=3 for each group,**P<0.01,***P<0.001. Fig. S7. Confocal analysis of the U87 cells treated with Au-DOX@PO for 6 hours.

Abstract: Additional file 2: Figure S2. Identification of SCAP-Exo. The morphology of SCAP-Exo was observed under TEM. The sizes and concentrations of SCAP-Exo were measured by nanoparticle tracking analysis. Western blot analysis showed that the exosomal surface markers Alix, CD9, and CD63 were expressed in SCAP-Exo, while calnexin was not expressed.

Abstract: Additional file 1: Fig. S1. Concentration of exosome. A, B: Concentration of exosomes as detected by Nanoparticle Tracking Analysis (NTA). Fig.S1A represents three NPC samples, while Fig.S1B represents three normal samples

Abstract: Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing, microarrays and can be utilized in functional in vitro/in vivo studies.

Abstract: Introduction: Viruses, extracellular vesicles (EVs) and endogenous retroviruses (ERVs) are types of sub-micron particles which are known to be released from a vast range of cell types, across many species. There are many medically relevant sub-micron particles which can enter healthy cells and enable the intercellular delivery of functional host-derived and foreign products, through their enclosed lipid layers. While multiple particle subsets have been identified, many of the properties, behaviors and biochemical functions have not been fully described and have yet to be characterized. Materials and Methods: CD4⁺ naïve T-cells were isolated from female C57BL6/N mice and stimulated with varying concentrations of PMA/I. In addition to concentration, the length of PMA/I activation was assessed. Supernatants and cells were harvested, filtered, and stained to be subsequently analyzed by Nanoscale Flow Cytometry, Nanoparticle Tracking Analysis and Flow Cytometry. Particle populations were quantified and sorted by size, by NTA. Labelling dye CFSE was used in conjunction with fluorescently conjugated CD81 and CD9 antibodies to separate EVs, including exosomes, from background signal. Naïve T-cell purity, viability and levels of activation were assessed by flow cytometry using CD3, CD4 and CD62L antibodies and viability staining. Results: Increasing PMA concentration led to a global increase in particles by T-cells and a specific increase in smaller particle production and were demonstrated to be significant by Welch’s T-test, when compared to non-activated and DMSO controls (p<0.0001). In addition to concentration, activation length also correlated with increases in total particle counts and a specific increase in the secretion of smaller particles in comparison to non-activated and DMSO controls (p<0.0001). Labelling techniques by NFC revealed an increased presence of CFSE-CD81 positive and CFSE-CD9 positive particles secreted by T-cells, treated for 24 hours, compared to the 0- and 12-hour timepoints. Conclusion: This work demonstrates preliminary steps and outlines methods to begin assessing discrete particle populations and subsets secreted by murine naïve T-cells. Being able to identify patterns of particle secretions by naïve T-cells, especially under immune-stimulated conditions, may be the solution to uncovering the necessary information on EV physiology, that is required to understand the roles EVs play in pathology and how these conserved pathways may lead conditions to become exacerbated. This knowledge is essential to uncovering the roles EVs play in pathophysiology, and in the development of novel rapid diagnostic tests, to screen for cancers, infections, autoimmune disorders, and numerous other pathological conditions.