Chapter 5 128 EVs released during NMP express predominantly CD81 Following the identification of EVs on the basis of tetraspanin expression, we examined the tetraspanin distribution on the released EVs by staining the NMP samples with CFDA-SE and one of the individual components of the antitetraspanin antibody mixture at a concentration equal to that used within the mixture. We observed that CD81+ EVs represented ~ 86% and ~74% of single and double-positive fluorescent events, respectively, across the time points analyzed (normalized average of time points 60, 180 and 360 minutes, Figure 3A-B). CD9+ and CD63+ EV were found to represent ~5% and ~9% of the detected single positive, and 16% and 9% of the double-positive fluorescent events, respectively. These findings show that tetraspanin CD81 is predominantly expressed on EVs released during NMP. Additionally, CFSE fluorescence was detected in conjunction with all tetraspanins studied, indicating that esterase activity is not exclusively linked to any of these tetraspanins. Leukocyte and Endothelial-derived EVs are released during NMP Surface proteins on EVs reflect the biological origin of their parental cells and next we determined the expression of either CD45 (as pan-leukocyte marker) or CD31 (as prominent endothelial marker) on the CD81+ EVs. We performed double staining of the perfusates with anti-CD45 or anti-CD31 in combination with anti-CD81, and analyzed each fluorescent population. For the CD45 single-positive events, ~52% of the events were still present after detergent treatment (data not shown) and no significant increases were observed during NMP when compared to baseline perfusates (T0 – 2.7E7 ± 5.4E6 objects/mL) despite high specificity of the mAbs as indicated by isotype controls (dashed lines - 1.7E5 ± 9.8E4 objects/mL, Figure 3C). Thus, CD45 single-positive EVs could not be discriminated from baseline perfusate signals. Analysis of CD81 and CD45 double-positive events yielded ~97.5% reduction after detergent treatment, a significant 18 / 19 / 23 – fold difference in objects/mL at 60 / 180 / 360 minutes of NMP compared to T0 (2.6E6 ± 1.4E5 objects/mL), and high specificity as indicated by isotype controls (4.3E4 ± 4.1E4 objects/mL, Figure 3C). Analysis of CD31 single-positive events showed ~91% reduction after detergent treatment, 11 / 14 / 13 – fold difference in objects/mL at each time point of sample drawing compared to T0 (2.6E5 ± 1.8E4 objects/mL), and an isotype background of
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