An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 39 Analysis of double-positive events revealed 5.96E7 ± 3.69E7 objects/mL total doublepositive particles across the 5 PPP samples measured, with a very limited number of artifact particles present after detergent treatment: 3.47E6 ± 4.48E6 objects/mL (~6% of total acquired events). This revealed that almost all double-positive particles measured (5.61E7 ± 3.36E7 objects/mL, ~94% of the total concentration before detergent treatment), were structurally intact, esterase-containing EV displaying common EV protein signatures in the form of tetraspanin markers (Figure 3e). By treating our samples with detergent we were able to identify to what extend our protocol discriminates legit EV signals from artefact signals. We observed that double-positive events were largely comprised of true EVs whereas the singlepositive populations showed a high degree of fluorescent particles still present after detergent treatment. Therefore, we concluded that the colocalization of two fluorophores (found as double-positive events before detergent treatment) represent CFSE+/Tetraspanin+ EV.
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