Chapter 3 46 Figure 6 – Tetraspanin distribution within 5 PPP samples. All samples were stained with CFDA-SE and an anti-tetraspanin mixture or one of the anti-tetraspanin antibodies at a concentration equal to that used in the mixture. a) Tetraspanin distribution determined using anti-CD9 [HI9a], anti-CD63 [H5C5] and antiCD81 [5A6], and b) their relative frequencies of double-positive events compared to that obtained with the anti-tetraspanin mixture. Results shown represent events (double-positive objects/mL) obtained with each of these staining combinations and are colored as follows: gray boxes – anti-tetraspanin mixture, orange boxes – anti-CD9, blue boxes – anti-CD63, green boxes – anti-CD81. Red dots: means of sample spread. Black dots, individual PPP samples. Colocalization of fluorophores indicates true EVs So far, the identification and discrimination of single EV from contaminating agents such as lipoproteins in PPP samples has been based on the notion that lipoproteins do not contain esterases, and hence cannot become fluorescently labelled by CFSE. However, as not all EV may contain esterases the quantification of double-positive events (CFSE+/Tetraspanin+) likely represents an underrepresentation when it comes to total EV. An alternative approach to the identification of single EV in PPP samples on the basis of intravesicular esterases would be the staining of samples with monoclonal antibodies (mAbs) targeting EV surface proteins. Based on the results presented in Figure 6b, we used anti-CD9 [clone HI9a] as this antibody was shown to recapitulate the majority of the tetraspanin signal. Anti-CD31 [clone WM59] was chosen as a secondary marker since CD31 is ubiquitously expressed within the vasculature and on diverse immune cell types, and therefore likely to be highly prevalent on EV in PPP.
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