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Extracellular vesicles (EVs) are mediators of intercellular communication, recently recognised for their clinical applications. Accurate characterisation and quantification of EVs are critical for understanding of their function and clinical relevance. Many platforms utilise fluorescence for EV characterisation, frequently labelling surface proteins to identify EVs. The heterogeneity of EVs and the lack of a universal protein marker encourages the use of generic EV labelling methods, including membrane labelling. Using nano‐flow cytometry, we evaluated six membrane dyes, including MemGlow and CellMask. Evaluation criteria included EV labelling efficacy, non‐specific labelling of very low‐density lipoproteins (VLDLs), brightness and dye aggregation. Significant variation was observed in dye performance, with certain dyes showing poor EV labelling efficacy or high affinity to VLDLs. Importantly, several promising candidates were identified for further investigation. Overall, this study highlights the importance of selecting appropriate membrane dyes for EV staining tailored to the aims of the study and the EV origin. MemGlow and CellMask proved favourable, allowing bright, sensitive staining of EV membranes with minimal aggregation. However, MemGlow showed an affinity to VLDLs, and CellMask requires additional sample handling for optimal labelling. These results contribute to deepening our understanding of EV membrane dyes, allowing for better dye selection and EV identification in future studies. [ABSTRACT FROM AUTHOR]

Skeletal muscle (SM) acts as a secretory organ, capable of releasing myokines and extracellular vesicles (SM‐EVs) that impact myogenesis and homeostasis. While age‐related changes have been previously reported in murine SM‐EVs, no study has comprehensively profiled SM‐EV in human models. To this end, we provide the first comprehensive comparison of SM‐EVs from young and old human primary skeletal muscle cells (HPMCs) to map changes associated with SM ageing. HPMCs, isolated from young (24 ± 1.7 years old) and older (69 ± 2.6 years old) participants, were immunomagnetically sorted based on the presence of the myogenic marker CD56 (N‐CAM) and cultured as pure (100% CD56+) or mixed populations (MP: 90% CD56+). SM‐EVs were isolated using an optimised protocol combining ultrafiltration and size exclusion chromatography (UF + SEC) and their biological content was extensively characterised using Raman spectroscopy (RS) and liquid chromatography mass spectrometry (LC‐MS). Minimal variations in basic EV parameters (particle number, size, protein markers) were observed between young and old populations. However, biochemical fingerprinting by RS highlighted increased protein (amide I), lipid (phospholipids and phosphatidylcholine) and hypoxanthine signatures for older SM‐EVs. Through LC‐MS, we identified 84 shared proteins with functions principally related to cell homeostasis, muscle maintenance and transcriptional regulation. Significantly, SM‐EVs from older participants were comparatively enriched in proteins involved in oxidative stress and DNA/RNA mutagenesis, such as E3 ubiquitin‐protein ligase TTC3 (TTC3), little elongation complex subunit 1 (ICE1) and Acetyl‐CoA carboxylase 1 (ACACA). These data suggest SM‐EVs could provide an alternative pathway for homeostasis and detoxification during SM ageing. [ABSTRACT FROM AUTHOR]

In this paper I will first describe how commodities are ‘promiscuously’ displayed by homeless queer youth in San Francisco in their attempts to pass as not only normal, but affluent. I will argue that when a youth successfully displays the signs of these commodities, they become part of a prosthetic shield that wraps around him or her. These shields protect homeless queer youth from the status degradation inherent in being classified as homeless. Thereby, homeless queer youth use the sign of the commodity to prevent being continually marked by spoiled identities. Their struggles reveal contradictions in the differential allocation of citizenship in which those who pass as ‘normal’ are granted the right to consume public space unmolested. I will conclude by suggesting that there has been a primarily unobserved convergence of neoliberal social policies within both San Francisco and gay politics detrimental to poor and otherwise marginal people.

The interview study reported here sought to identify the perceived trajectory of romantic relationships of a cohort of Oakland African American gay/bisexual adolescents. Biographical interviews were used to identify cultural models of romantic relationships in the study sample and discovered a trajectory of four phases. In the antecedent to the trajectory, the youth enters the gay/bisexual social environment. In the first phase, the youth meets a male partner. In the second phase, the youth "talks to" the partner over a period of days or weeks. In the third phase, the couple enters into a "boyfriend" relationship and initiates sex. In the fourth phase, the couple experiences conflicts over infidelity and breaks up. As a consequence, the gay/bisexual youth experiences an aftermath that includes low self-esteem, trying to get back with the partner, and revenge sex. Each phase is described with informant quotations. Some implications for HIV prevention with these adolescents are discussed. (Contains 2 tables and 1 figure.)

Current state-of-the-art tools for analysing extracellular vesicles (EVs) offer either highly sensitive but unidimensional bulk measurements of EV components, or high-resolution multiparametric single-particle analyses which lack standardization and appropriate reference materials. This limits the accuracy of the assessment of marker abundance and overall marker distribution amongst individual EVs, and finally, the understanding of true EV heterogeneity. In this study, we aimed to define the standardized operating procedures and reference material for fluorescent characterization of EVs with two commonly used EV analytical platforms-nanoparticle tracking analysis (NTA) and nano-flow cytometry (nFCM). We achieved quantitative fluorescence analyses on ZetaView NTA and NanoAnalyzer nFCM instruments, by utilizing yellow-green FluoSpheres (FS) with assigned ERF (equivalent reference fluorophore) values. This standardization technique allowed for fluorescent EV signal to be expressed in ERF units (indicative of bound fluorescent antibodies per EV), thus enabling measurement of target protein marker abundance on individual EVs, and in the whole EV population. The NTA's and nFCM's limits of detection (LoD) were evaluated at 21 and 9 Alexa Fluor 488 (AF488) molecules, respectively. To complement the limited quantification of markers expressed in a few copies per single EV, in-line bulk fluorescence measurements with a plate reader were performed. This provided absolute marker quantification and more insightful analyses of EV heterogeneity and marker stoichiometry. The standardization method outlined in this work unlocks the full analytical potential of NTA and nFCM, enabling cross-platform data comparison. At the same time, it highlights some of the technical challenges and considerations and thus contributes to the ongoing efforts towards the development of EV analytical tools., Competing Interests: D.M. is an Applied Scientist in the company HansaBioMed Life Sciences, which is the manufacturer of the products (PURE‐EVs, FLuoEVs, lyophilized exosomes and microvesicles standards, ExoTEST ELISA kits) used in this study. J.B. is a Junior Researcher and B.P. is the Head of Research in the company NanoFCM, which is the manufacturer of the NanoAnalyzer U30 instrument used in this study., (© 2025 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)