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. 2019 Mar 5;8(1):1585163. doi: 10.1080/20013078.2019.1585163

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© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Confirmation of the presence of the pro-coagulant factors TF and phosphatidylserine (PS) on EVs. (a) Detection of TF⁺-EVs in conditioned cell culture media using bead-based flow cytometry with beads coated for different tetraspanins, n = 7. (b) TF expression on isolated EVs (10⁸ particles/ml) determined by bead-based flow cytometry with anti-CD63/CD81/CD9-coated beads, n = 6. (c) TF activity of EV isolates obtained by UF-SEC (10⁸ particles/ml), volume-matched protein isolates obtained by UF-SEC and volume-matched sham EV isolated from unconditioned cell culture media, n = 5. (d) Percentage of TF⁺ cells (the gate was set so that 98% of the cells stained with the isotype control were negative), n = 4. (e) Detection of PS⁺-EVs in conditioned cell culture media using bead-based flow cytometry with beads coated for different tetraspanins, n = 7. (f) PS expression on isolated EVs (10⁸ particles/ml) determined by bead-based flow cytometry with anti-CD63/CD81/CD9-coated beads, n = 9. (g) Thrombin generation as determined by prothrombinase assay in the absence or presence of the PS blocker annexin V; n = 4. (h) PS externalization on control and CSE-exposed BEAS-2B cells as determined by flow cytometry using annexin V and PI staining; n = 6. *p < 0.05; AnxV: annexin V; CSE: cigarette smoke extract; ctrl: control; EVs: extracellular vesicles; FC: fold-change; n.s. : not significant; PI: propidium iodide; PS: phosphatidylserine; RFU: relative fluorescent units; TF: tissue factor; TRPS: tuneable resistive pulse sensing; UCM: unconditioned medium isolate.