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. 2020 Nov 18;21(22):8723. doi: 10.3390/ijms21228723

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Isolated large EVs can be triple-labelled and individual events can be scrutinised post-acquisition. (A) The proportion of CMO+ events expressing a single EV marker varied across replicates. The percentage positive events within the CMO+ MESF > 50 gate which expressed either no EV marker (CMO only), CD63, CD9, or LAMP1 varied across replicates. Black bar represents the mean across 10 (CMO or CD63) or 5 (CD9 or LAMP1) replicates. CMO only 63.3% (± 4.6 SEM), CD63 only 19% (± 2.2 SEM), CD9 (± 0.5 SEM) only 10.3% and LAMP1 only 47.6% (± 3.8 SEM). (B) UW228-2 cell derived EVs differentially express EV markers CD63 and CD9, or both. EVs were labelled with CMO, CD63 BV421 and CD9 AF647. CMO ⁺ events (MESF > 50) were analysed for co-expression with CD63, CD9 or both. Quadrant gating of bivariate plots for fluorescence intensity in channel 07 (BV421) and channel 11 (AF647) demonstrated single labelled (CMO⁺ only), dual and triple labelled EV populations (i). Percentage positive in each quadrant is shown. Representative experiment. Gallery images display examples of individual events from each of the 4 quadrants (ii). Bright Field (BF) and side scatter (SSC) channels are shown alongside CMO, CD63 BV421 and CD9 AF647. Of the CMO+ MESF >50: 75.1% (± 3.0 SEM) were CMO ⁺ only; 13.3% (± 2.4 SEM) expressed CD63 BV421 and 3.5% (± 0.8 SEM) expressed CD9 AF647. 7.1% (± 1.1 SEM) of the CMO⁺ events expressed both CD63 and CD9. (C) UW228-2 cell derived EVs differentially express EV markers CD63 and LAMP1, or both. EVs were labelled with CMO, CD63 BV421 and LAMP1 AF647. CMO⁺ events were analysed for co-expression with CD63, LAMP1 or both. Quadrant gating of bivariate plots for fluorescence intensity in channel 07 (BV421) and channel 11 (AF647) demonstrated single labelled (CMO⁺ only), dual and triple labelled EV populations (i). Percentage positive in each quadrant is shown. Representative experiment. Gallery images display examples of individual events from each of the 4 quadrants (ii). Bright Field (BF) and side scatter (SSC) channels are shown alongside CMO, CD63 BV421 and LAMP1 AF647. Of the CMO+ MESF >50: 54.1% (± 4.3 SEM) were CMO ⁺ only; 2.8% (± 0.4 SEM) expressed CD63 BV421 and 25.6% (± 2.7 SEM) expressed LAMP1 AF647 whilst 18.0% (± 2.8 SEM) of the CMO ⁺ events expressed both CD63 and LAMP1. (* p = 0.0159 or 0.0317; ** p = 0.0079). Mann Whitney U test.