Chapter 6 158 Figure 3 – Quantification of CD9+HLA-A3+ events in mixed PPP samples. A) Serial dilutions (2-fold) of 5 double-stained (anti-CD9 and anti-HLA-A3) matched donor-recipient samples shows that our setup is able to discriminate CD9+HLA-A3+ EVs above recipient ‘self’ background (HLA-A3- recipient signals, indicated by the green dashed lines) down to ~1%. B) Coincidence testing through serial dilution. A linear correlation between the concentrations of CD9+HLA-A3+ EVs and dilution factor (range: 100% to 1.56% HLA-A3+ PPP) for each serial dilution experiment confirmed single particle analysis in mixed PPP samples. C) Stable (standardized) fluorescent signals for both detection channels (Ch01 – HLA-A3 / Ch05 – CD9) were observed irrespective of the dilution factor.
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