Journal of Extracellular Vesicles最新文献

{"title":"Correction to “Synthetic Biology-Based Bacterial Extracellular Vesicles Displaying BMP-2 and CXCR4 to Ameliorate Osteoporosis”","authors":"","doi":"10.1002/jev2.70095","DOIUrl":"https://doi.org/10.1002/jev2.70095","url":null,"abstract":"<p>H. Liu, P. Song, H. Zhang, et al., “Synthetic Biology-Based Bacterial Extracellular Vesicles Displaying BMP-2 and CXCR4 to Ameliorate Osteoporosis,” <i>Journal of Extracellular Vesicles</i> 13 (2024): e12429, https://doi.org/10.1002/jev2.12429.</p><p>In Table 1 of the originally published article, the Characteristic of ECN, “Escherichia coli Nissle 1917,” is incorrect. This should read “<i>Escherichia coli</i> Nissle 1917 carrying the DE3 phage encoding for T7-RNA Polymerase.”</p><p>In addition, Figure 2g in the originally published article is incorrect. The correct figure is shown below.</p><p></p><p>We apologize for this error.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 6","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70095","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144190950","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reflecting on 2100 Days at the Helm of Journal of Extracellular Vesicles: A Thank You Editorial","authors":"Jan Lötvall","doi":"10.1002/jev2.70096","DOIUrl":"https://doi.org/10.1002/jev2.70096","url":null,"abstract":"&lt;p&gt;As I have stepped down as Editor-in-Chief of the &lt;i&gt;Journal of Extracellular Vesicles (JEV)&lt;/i&gt; on April 30th, 2025, exactly 2100 days after assuming the role on August 1st, 2019, I find myself reflecting on the extraordinary journey of growth and increased understanding of EV biology, that I have had the honour to share with many others within the extracellular vesicle (EV) community. Leading this journal has been one of the most rewarding experiences of my scientific career, and I am deeply grateful to the authors, the editorial team, reviewers and our readers who have contributed to its growth and success.&lt;/p&gt;&lt;p&gt;When I took on this role, the field of EV research, and &lt;i&gt;JEV&lt;/i&gt;, was in a rapidly expanding phase, and the journal had just months prior received its first impact factor. However, during the past 5 years, the growth of the field and the journal is further accelerating, and we have experienced a deepened understanding of EV biology, in parallel with technological advancements in both the isolation and characterisation of EVs. &lt;i&gt;JEV&lt;/i&gt; has, from its first inception in 2012, been at the forefront of these developments, publishing groundbreaking studies that have shaped the discourse and pushed the boundaries of our discipline, and is still on a rapidly growing trajectory.&lt;/p&gt;&lt;p&gt;Employing Sarah has, without question, been the most important improvement in the journal's management in the last 5 years, as she professionally manages and oversees the journal, far beyond the scope of what part-time Editors are capable of. Thank you for joining JEV Sarah!&lt;/p&gt;&lt;p&gt;The journal would not be what it is without the dedication of our editorial team, the rigor of our peer reviewers, as well as the trust of the scientific community that submit their work to the journal. Before the employment of Sarah Williams, I have for some periods had the administrative editorial support of several well-established researchers in the field, including Cecilia Lässer and Deborah Goberdhan. In addition, the crucial role of the Deputy Editors as well as Associate Editors in further scrutinising submissions, is essential for any journal. During my tenure, I have had the support of multiple Deputy Editors, including Simon Powis, Esther Nolte-‘t Hoen, Hubert Yin, Mary Bebawy, Michael Freeman, Roosmarijn Vandenbroucke, Yong Song Gho, An Hendrix, and recently also Cherie Blenkrion, Simon Swift, Owen Davis and Dolores Di Vizio, and a broad group of Associate Editors. As EiC, I implemented a multi-step approach to assessing manuscripts, requiring at least two pairs of eyes evaluating a manuscript before the journal declines consideration of a submission, and multiple other assessments by Editors and Reviewers before any work is found acceptable for publication. To retain a reasonable and fair assessment of submissions, I have worked under the principle that one person should be at the helm of the journal, implementing one clear editorial vision for which work is suitable ","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 6","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70096","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144190949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Glutamatergic Regulation of miRNA-Containing Intraluminal Vesicle Trafficking and Extracellular Vesicle Secretion From Cortical Neurons","authors":"Marcela Bertolio,&nbsp;Qiyi Li,&nbsp;Francesca E. Mowry,&nbsp;Kathryn E. Reynolds,&nbsp;Rashed Alananzeh,&nbsp;Haichao Wei,&nbsp;Kyoeun Keum,&nbsp;Rachel Jarvis,&nbsp;Jiaqian Wu,&nbsp;Yongjie Yang","doi":"10.1002/jev2.70100","DOIUrl":"https://doi.org/10.1002/jev2.70100","url":null,"abstract":"<p>Neuronal extracellular vesicles (microvesicles and exosomes) are emerging secreted vesicular signals that play important roles in the CNS. Currently, little is known about how glutamatergic signalling affects the subcellular localisation of exosome precursor intraluminal vesicles (ILVs), microRNA (miR) packaging into ILVs and in vivo spreading of neuronal EVs. By selectively labelling ILVs and exosomes (but not plasma membrane-derived MVs) with GFP-tagged human CD63 (hCD63-GFP) in cortical neurons, we found that glutamate stimulation significantly redistributes subcellular localisation of hCD63-GFP⁺ ILVs, especially decreasing its co-localisation with multi-vesicular body (MVB) marker Rab7 while substantially promoting EV secretion. Interestingly, glutamate stimulation only modestly alters EV miR profiles based on small RNA sequencing. Subsequent in vivo cortical neuronal DREADD activation leads to significantly more widespread hCD63-GFP⁺ area in hCD63-GFP^f/+ mice, consistently supporting the stimulatory effect of glutamatergic activation on neuronal EV secretion and spreading. Moreover, in situ localisation of hCD63-GFP⁺ ILVs and hCD63-GFP⁺ secreted exosomes from specialised HB9⁺ and DAT⁺ neurons were also illustrated in the CNS. Taken together, our results demonstrated that glutamate activity stimulates neuronal exosome secretion and spreading in vitro and in vivo, but only modestly affects miR cargo packaging in neuronal exosomes.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 6","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70100","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144171946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Recombinant Outer Membrane Vesicles (OMVs)-Based Vaccine Against Helicobacter pylori Infection in Mice","authors":"Qiong Liu,&nbsp;Biaoxian Li,&nbsp;Jinrong Ma,&nbsp;Xiao Lei,&nbsp;Junpeng Ma,&nbsp;Yanyan Da,&nbsp;Zhiyong Zhou,&nbsp;Jiaqi Tao,&nbsp;Xinyi Ren,&nbsp;Ting Zeng,&nbsp;Zhiting Xie,&nbsp;Haiyan Lin,&nbsp;Zihui Jin,&nbsp;Yi Wan,&nbsp;Liang Zhang,&nbsp;Donglin Lai,&nbsp;Yaping Guo,&nbsp;Jing Li,&nbsp;Yinpan Shang,&nbsp;Lu Shen,&nbsp;Ziwei Tao,&nbsp;Tian Gong,&nbsp;Chengsheng Zhang","doi":"10.1002/jev2.70085","DOIUrl":"https://doi.org/10.1002/jev2.70085","url":null,"abstract":"<p>The current vaccine development for <i>Helicobacter pylori</i> (<i>H. pylori</i>) still faces challenges of weak immune responses stimulated by existing antigens and a lack of safe adjuvants. The modification of the lipopolysaccharide (LPS) structure by <i>H. pylori</i> is an important mechanism involved in its immune escape. In this study, we developed a novel recombinant vaccine candidate against <i>H. pylori</i> infection by knocking down the key genes (lpxE, lpxF and futB) of LPS modification and employing the bacterial outer membrane vesicles (OMVs) as a vector for delivering UreB, VacA and CagA antigens, and then evaluated its safety and immune protective efficacy in vitro and in vivo mouse model. We measured the antibody and cytokine productions, detected the subtypes of immune cells, and examined the histopathological changes in mice from the control and various experimental groups. We revealed that this OMV-based recombinant vaccine candidate could induce specific humoral immune responses and a Th1/Th2/Th17 mixed immune response, with Th17 being predominant, and markedly protect the mice from <i>H. pylori</i> infection. Our findings suggest that the OMVs with the genetically engineered LPS may function as a vector for delivering recombinant antigens and safe adjuvants for the development of novel vaccine candidates against <i>H. pylori</i> infection.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 5","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70085","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hemagglutinin Protease HapA Associated With Vibrio cholerae Outer Membrane Vesicles (OMVs) Disrupts Tight and Adherens Junctions","authors":"Palwasha Baryalai,&nbsp;David Irenaeus,&nbsp;Eric Toh,&nbsp;Madeleine Ramstedt,&nbsp;Bernt Eric Uhlin,&nbsp;Aftab Nadeem,&nbsp;Sun Nyunt Wai","doi":"10.1002/jev2.70092","DOIUrl":"https://doi.org/10.1002/jev2.70092","url":null,"abstract":"<p>This study explores the virulence mechanisms of <i>Vibrio cholerae</i>, with a particular emphasis on HapA, a zinc metalloprotease delivered via outer membrane vesicles (OMVs). The findings reveal that OMV-associated HapA disrupts the integrity of tight and adherens junctions in intestinal epithelial cell models more effectively than its purified counterpart, suggesting that association with OMVs substantially potentiates the pathogenic effects of HapA. The study further details the uptake of <i>V. cholerae</i> OMVs by epithelial cells, as well as their targeted degradation of key junctional proteins, including claudin, ZO-1, and β-catenin. These results highlight the critical role of OMV-associated HapA in compromising epithelial barrier function. Additionally, the use of spheroids and intestinal organoids in our experiments provides deeper insight into bacterial pathogenesis, offering valuable information for the development of targeted therapeutic strategies.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 5","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70092","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144135788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CD24 Regulates the Formation of Ectosomes in B Lymphocytes","authors":"Hong-Dien Phan,&nbsp;Kaitlyn E. Mayne,&nbsp;Willow R. B. Squires,&nbsp;Grant R. Kelly,&nbsp;Reilly H. Smith,&nbsp;Rashid Jafardoust,&nbsp;Sherri L. Christian","doi":"10.1002/jev2.70093","DOIUrl":"https://doi.org/10.1002/jev2.70093","url":null,"abstract":"<p>CD24 is a glycophosphatidylinositol-linked protein that regulates B cell development. We previously reported that stimulation of CD24 on donor B cells promotes the transfer of functional receptors to recipient B cells via extracellular vesicles (EVs). However, the mechanisms regulating CD24-mediated formation of bioactive EVs are unknown. Using bioinformatics, we found a connection between CD24, and PI3K/AKT, tran and mTOR. To determine if these pathways regulate EV release, we used flow cytometry to follow the transfer of EVs carrying lipid-associated GFP and surface IgM from donor to recipient B cells. Using chemical and genetic inhibition, we found that a PI3K/mTORC2/ROCK/actin pathway regulates bioactive EV formation via activation of acid sphingomyelinase (aSMase) upstream of PI3K. Using single EV analysis, we found that CD24 regulates the formation of the subset of bioactive EVs that are taken up by recipient cells and not total EVs. Interestingly, we also found that ROCK and aSMase modulate ectosome but not exosome formation, when CD24 is stimulated. Lastly, through live cell imaging, we found that PI3K and ROCK are required for inducing membrane dynamics associated with EV formation. These data suggest that this pathway regulates bioactive EV release that, in turn, could regulate B cell development.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 5","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70093","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144135740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Extracellular Vesicle (EV) Targeted Cells Release Secondary Effector EVs: Indication of How To Account for Histocompatibility and Disease Specificity of EV Treatments","authors":"Philip W. Askenase","doi":"10.1002/jev2.70076","DOIUrl":"https://doi.org/10.1002/jev2.70076","url":null,"abstract":"<p>The central hypothesis presented here is that released extracellular vesicles (EVs) can act primarily on targeted cells to induce the production of secondary EVs to mediate the final biological events. Compared here are different instances. In one, EVs, primarily produced by CD8⁺ suppressor T cells, are activated in immune tolerance. These EVs transfer to companion recipient macrophages (Macs) the ability to generate production of secondary inhibitory EVs that affect the final-acting effector T cells. In a second instance of treating spinal cord injury (SCI), primary-acting mesenchymal stromal cell (MSC)-derived EVs target local tissue M2-type Macs to release secondary EVs that subsequently affect the local neuro microvasculature to mediate healing. Thus, these are very different systems acting similarly in this way. Per treatments with Mesenchymal Stromal Cells (MSCs), our proposal explains how their released EVs can act across tissue histocompatibility barriers and exhibit a seeming “disease specificity,” resulting in the healing of many diverse injuries and a wide variety of pathologic conditions. It is postulated that the recipients of the primary EVs, the secondarily acting cells, are often but not exclusively Macs. These are among the local responding secondary-acting cells that produce transplantation-matched EVs. Further, the secondary-acting MSC-derived primary EVs that are clinically active in many diverse instances led to the additional hypothesis that secondary EVs produced by targeted local cells may be appropriate to each specific instance to explain such disease specificity. We propose that there may be many other examples to be uncovered in which primary EVs similarly induce secondary EV healing effects.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 5","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70076","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144135787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Arabidopsis Produces Distinct Subpopulations of Extracellular Vesicles That Respond Differentially to Biotic Stress, Altering Growth and Infectivity of a Fungal Pathogen","authors":"Benjamin L. Koch,&nbsp;Brian D. Rutter,&nbsp;M. Lucía Borniego,&nbsp;Meenu Singla-Rastogi,&nbsp;Dillon M. Gardner,&nbsp;Roger W. Innes","doi":"10.1002/jev2.70090","DOIUrl":"https://doi.org/10.1002/jev2.70090","url":null,"abstract":"<p>Extracellular vesicles (EVs) secreted by mammalian cells are highly heterogeneous in content and function. Whether this is also true for EVs secreted by plant cells is not yet known. To address this, we used high-resolution density gradient ultracentrifugation and total internal fluorescence microscopy (TIRF-M) to purify and distinguish distinct subpopulations of Arabidopsis EVs. The EV marker protein TETRASPANIN 8 (TET8) was detected specifically in medium-density EVs. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted in mostly separate EV populations using TIRF-M, while PEN1 was co-secreted with PENETRATION 3 (PEN3) much more often. Secretion of EV subpopulations marked by TET8, PEN1 and RPM1-INTERACTING PROTEIN 4 (RIN4) into the apoplast and onto the leaf surface was induced by phytohormones, changes in temperature and infection with fungal pathogens. Treatment of Arabidopsis seedlings with plant EVs delayed the progression of fungal infection by altering fungal germ tube development and fungal morphology. Significantly, extracellular RNAs, including miRNAs and siRNAs, did not co-fractionate with TET8-labeled EVs, and instead, co-fractionated with extravesicular ARGONAUTE proteins in high-density fractions. Together, these data indicate that Arabidopsis EVs are highly heterogeneous and contribute to immunity but are unlikely to mediate cross-kingdom RNA interference.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 5","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70090","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144135741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Extracellular Vesicle-Mediated Delivery of 20S Proteasomes Enhances Tau Degradation in Recipient Cells","authors":"Jiseong Kim,&nbsp;Yuping Zhao,&nbsp;Hyun Young Kim,&nbsp;Sumin Kim,&nbsp;Yanxialei Jiang,&nbsp;Min Jae Lee","doi":"10.1002/jev2.70086","DOIUrl":"https://doi.org/10.1002/jev2.70086","url":null,"abstract":"<p>The 26S proteasome holoenzyme comprises 20S catalytic and 19S regulatory complexes. Accumulating evidence suggests that the majority of proteasomes in the extracellular space exist as free 20S proteasomes; however, their origin and pathophysiological function remain to be determined. Here, we report that cellular proteasomes are effectively packaged into the lumen of extracellular vesicles (EVs) and secreted in a structurally intact and enzymatically active 20S form. We further demonstrate that EV-encapsulated 20S proteasomes are delivered to recipient cells and facilitate the degradation of overexpressed tau proteins without disrupting global proteolytic pathways. These findings highlight a novel cell-to-cell communication system that transports the proteasomes to target cells for the clearance of proteotoxic substrates. Further characterisation of this homeostatic mechanism will improve our understanding of organismal stress response mechanisms and may provide a therapeutic approach to treat various proteinopathies, including Alzheimer's disease.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 5","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70086","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144085478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Non-Specific Particle Formation During Extracellular Vesicle Labelling With the Lipophilic Membrane Dye PKH26","authors":"Laurel A. Haines,&nbsp;Alex A. Baeckler,&nbsp;Sophi J. Schofield,&nbsp;Eric P. Palmer,&nbsp;Bradley F. Guilliams,&nbsp;Melinda A. Meyers,&nbsp;Daniel P. Regan","doi":"10.1002/jev2.70079","DOIUrl":"https://doi.org/10.1002/jev2.70079","url":null,"abstract":"<p>Current approaches for the fluorescent labelling of extracellular vesicles (EVs) have been reported to produce widely variable and controversial results, highlighting a significant need for validated, reproducible labelling methods to advance the field of EV research. Lipophilic membrane dyes are commonly used but have been shown to produce non-specific fluorescent particles that are indistinguishable from labelled EVs, confounding experimental results. We aimed to distinguish conditions that can either promote or reduce the formation of non-specific dye particles when using the prototypical lipophilic membrane dye PKH26. We optimised a labelling approach that minimises the production of non-specific dye particles by altering buffer conditions during staining and validated this method across cell-based and in vivo systems of EV biodistribution. To do this, we specifically isolated small EVs using ultrafiltration and size exclusion chromatography and validated sample purity and post-isolation processing steps. We then used single-EV spectral flow cytometry and transmission electron microscopy to investigate the impact of four different buffer conditions on PKH26 non-specific particle formation. We also determined the extent to which non-specific PKH26 particles were detectable in cell-based assays and in vivo within mouse lymph nodes using flow cytometry, immunofluorescence, and intravital imaging. By optimising buffer conditions to eliminate additional protein, we were able to minimise the formation of dye aggregates while maintaining efficient EV labelling, producing a much higher signal-to-noise ratio both in vitro and in vivo. We also demonstrate that failure to include proper vehicle controls can have significant implications on experimental results, leading to false positive data. This work emphasizes the importance of adequately benchmarking EV labelling approaches as it is essential for accurate evaluation of EV trafficking in physiologic and pathologic states.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"14 5","pages":""},"PeriodicalIF":15.5,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.70079","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144085477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}