Fig. 5

Phenotyping Strategy and enumeration of CNS-EEV from different patient populations. Diagram (A) and representative dot plots (B) of gating strategy to detect EV derived from CNS endothelial cells. EV of appropriate size were analyzed for the presence of CD3 or CD41 to determine if they were of lymphocyte or platelet origin, respectively. EV negative for CD3 and CD41 (CD3/CD41-) were analyzed for the presence of pan-endothelial markers CD31, CD105, and CD144. EV positive for CD31, CD105, or CD144 are referred to as EEV31, EEV105, and EEV144, respectively (Reviewed in Table 2). To determine if EEV31, EEV105, and EEV144 are derived from CNS endothelial cells, these events were analyzed for the expression of MAL via pETX-647 binding. EEV31, EEV105, and EEV144 positive for pETX/MAL are referred to as CNS-EEV31, CNS-EEV105, and CNS-EEV144, respectively (Reviewed in Table 2). Representative scatter plots of anti-CD-stained EV and their isotype controls are depicted in Additional file 1: Fig. S1. C Enumeration of CNS-EEV31, CNS-EEV105, and CNS-EEV144 per μL of plasma from different patient groups including HC, active RRMS patients not receiving DMT (Active), stable RRMS patients not receiving DMT (Stable), or stable RRMS patients receiving natalizumab (NTZ) or ocrelizumab (OCZ). Results are displayed as box and whisker plots with each dot representing an individual patient. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 determined by one-way ANOVA with post-hoc Tukey HSD Test. D Western blot analysis of EV lysates for CD31, CD105, and CD144. HUVEC lysate was used as controls