An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 43 We observed that the concentrations of double-positive events were linearly proportional to the dilution factor (Figure 5a) while the ERF of both fluorescent signals remained stable: mean 113.47 (range 55.07-157.55) for CFSE and mean 31.83 (range 28.2-36.8) for APC (Figure 5b), showing that the IFCM platform is capable of accurately quantifying individual EV. Serial dilution resulted into a larger spread of CFSE ERF values at lower dilutions (64x and 256x) only, which was interpreted to be a consequence of the lower number of particles analyzed. Additionally, doublepositive EV concentrations at the aforementioned dilutions came close to the previously established background of our assay (~E5 objects/mL). The observed linear reduction in concentration of double-positive events according to the dilution factor, and the stable ERF signals for both fluorescent markers, confirm that the IFCM platform is able to quantify true single EV. Additionally, we were able to verify that our gating strategy correctly identifies and selects single EV (by excluding multiplet events).
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