Chapter 5 130 Figure 3 – Phenotyping and determination of the concentration of EVs released during NMP. NMP samples were stained with CFDASE in combination with anti-CD9, anti-CD63 or anti-CD81, acquired using IFCM and analyzed using the previously described gating strategies. A) Tetraspanin profile of tetraspanin single-positive objects. B) Tetraspanin profile of double-positive objects. Additionally, perfusates were stained with anti-CD81 (predominantly expressed on EVs released during NMP) in combination with C) anti-CD45 (as pan-leukocyte marker), or D) CD31 (as prominent endothelial marker). Significantly increased levels of EVs were detected compared to T0 for all identified subpopulations (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), with the exception of CD45 single-positive EVs. Red dots/black lines representing the means/median values of each time point, respectively. Dashed lines: mean (green) ± standard deviation (darkred) of respective isotype controls (shown for C and D). E) Assessment of cellular origin of detected CD81+ EVs. For all time points and samples, the fraction of double-positive events compared to total detected CD81+ events (both single-positive and double-positive events) was calculated and averaged to determine the relative abundance of double-positive EVs for either population, thus allowing deduction of cellular origin.
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