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

Figure 4.

Figure 4

Optimised acquisition by Imaging flow cytometry and florescence membrane labelling can distinguish large extracellular vesicles from background and enable multiplex labelling. (A) Hierarchical gating can confidently refine the EV population for characterisation. Samples were acquired for 5 min using the ISX INSPIRE software and all events visualised using bivariate plots for fluorescence intensity in channel 03 (CMO) and channel 06 (side scatter). Initial gates for speed beads and CMO− events were set using the Acquisition buffer only and unstained EV samples included in each run (Gate i). These gates were applied to the labelled samples. Saturated events were excluded from the analysis by gating on the histogram plot: Raw Max Pixel for Channel 06 (Gate ii) thus removing the speed beads and very high side scatter events. An initial CMO + gate was placed (Gate iii). A lower fluorescence threshold of MESF > 50 in channel 3 (CMO) was set in each experiment using the regression analysis of Quantibrite PE Beads as described. The CMO + gate was anchored using co-ordinates for the equivalent fluorescence intensity and a further CMO + MESF > 50 gate applied to the histogram (Gate iv). Finally, a refined gate was used to eliminate outliers (Gate v). The analysis template was set for each experiment and applied to all samples. (B) Percentage positive CMO+ events (MESF > 50). The percentage of CMO+ events (MESF > 50) is represented by an orange dot for 10 replicate experiments. Black bar represents mean 18.73% (± 1.2 SEM). (C) A compensation matrix was applied to all samples. The compensation wizard was used to create a matrix which was applied to all samples. (D) Single, double, and triple labelling protocols were used for compensation and accurate gating. Dual labelled EVs provided Fluorescence minus one (FMO) controls for (i) AF647; EVs labelled with CMO and CD63 BV421) or (ii) BV421; (EVs labelled with CMO and LAMP1 AF647) and used to set positive gates. (E) UW228-2 cells were used to optimise multiplex labelling. Cell mask orange (CMO) provides a general membrane label (i) and could be used to co-label with EV markers CD63 BV421 and CD9 AF647 (ii) or CD63 BV421 and LAMP1 AF647 (iii). Representative gallery images from IDEAS software are shown.