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  1. Article ; Online: Preventing swarm detection in extracellular vesicle flow cytometry: a clinically applicable procedure.

    Buntsma, Naomi C / Shahsavari, Mona / Gąsecka, Aleksandra / Nieuwland, Rienk / van Leeuwen, Ton G / van der Pol, Edwin

    Research and practice in thrombosis and haemostasis

    2023  Volume 7, Issue 4, Page(s) 100171

    Abstract: Background: Flow cytometry is commonly used to detect cell-derived extracellular vesicles in body fluids such as blood plasma. However, continuous and simultaneous illumination of multiple particles at or below the detection limit may result in the ... ...

    Abstract Background: Flow cytometry is commonly used to detect cell-derived extracellular vesicles in body fluids such as blood plasma. However, continuous and simultaneous illumination of multiple particles at or below the detection limit may result in the detection of a single event. This phenomenon is called swarm detection and leads to incorrect particle concentration measurements. To prevent swarm detection, sample dilution is recommended. Since the concentration of particles differs between plasma samples, finding the optimal sample dilution requires dilution series of all samples, which is unfeasible in clinical routine.
    Objectives: Here we developed a practical procedure to find the optimal sample dilution of plasma for extracellular vesicle flow cytometry measurements in clinical research studies.
    Methods: Dilution series of 5 plasma samples were measured with flow cytometry (Apogee A60-Micro), triggered on side scatter. The total particle concentration between these plasma samples ranged from 2.5 × 10
    Results: Swarm detection was absent in plasma samples when diluted ≥1.1 × 10
    Conclusion: To prevent swarm detection in a series of clinical samples, the measurement count rate of a single diluted plasma sample can be used to determine the optimal dilution factor. For our samples, flow cytometer, and settings, the optimal dilution factor is ≥1.1 × 10
    Language English
    Publishing date 2023-05-02
    Publishing country United States
    Document type Journal Article
    ISSN 2475-0379
    ISSN (online) 2475-0379
    DOI 10.1016/j.rpth.2023.100171
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Single molecule network analysis identifies structural changes to caveolae and scaffolds due to mutation of the caveolin-1 scaffolding domain.

    Wong, Timothy H / Khater, Ismail M / Joshi, Bharat / Shahsavari, Mona / Hamarneh, Ghassan / Nabi, Ivan R

    Scientific reports

    2021  Volume 11, Issue 1, Page(s) 7810

    Abstract: Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three scaffold domains, hemispherical S2 scaffolds and smaller ... ...

    Abstract Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three scaffold domains, hemispherical S2 scaffolds and smaller S1B and S1A scaffolds. The caveolin scaffolding domain (CSD) is a highly conserved hydrophobic region that mediates interaction of CAV1 with multiple effector molecules. F92A/V94A mutation disrupts CSD function, however the structural impact of CSD mutation on caveolae or scaffolds remains unknown. Here, SMLM network analysis quantitatively shows that expression of the CAV1 CSD F92A/V94A mutant in CRISPR/Cas CAV1 knockout MDA-MB-231 breast cancer cells reduces the size and volume and enhances the elongation of caveolae and scaffold domains, with more pronounced effects on S2 and S1B scaffolds. Convex hull analysis of the outer surface of the CAV1 point clouds confirms the size reduction of CSD mutant CAV1 blobs and shows that CSD mutation reduces volume variation amongst S2 and S1B CAV1 blobs at increasing shrink values, that may reflect retraction of the CAV1 N-terminus towards the membrane, potentially preventing accessibility of the CSD. Detection of point mutation-induced changes to CAV1 domains highlights the utility of SMLM network analysis for mesoscale structural analysis of oligomers in their native environment.
    MeSH term(s) Caveolin 1/chemistry ; Cell Line ; Humans ; Mutation ; Protein Conformation ; Protein Domains/genetics
    Chemical Substances CAV1 protein, human ; Caveolin 1
    Language English
    Publishing date 2021-04-08
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/s41598-021-86770-6
    Database MEDical Literature Analysis and Retrieval System OnLINE

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