An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 37 Figure 2 – Gating strategy for the detection of single EV through exclusion of coincident events and fluorescent background analysis. a) Generalized concept. First, particles with SSC intensities ≤ 900 a.u. are selected, effectively selecting all (fluorescent) particles ≤ 400 nm (I). Subsequently, coincidence detection is carried out based on the number of fluorescent spots within the pixel grid determined with the standard intensity mask. Events showing 0 or 1 spot within each channel are selected and used in the subsequent analysis (II & III); events showing more than 1 spot are excluded from analysis. Lastly, the distance between the individual fluorescent spots on the different detection channels is calculated and events not overlapping on the pixel grid are excluded (IV). Visual examples of excluded events are shown below each graph. b) Representative example of unstained and single-stained PPP samples (stained with CFDASE or the anti-tetraspanin mixture -composed of anti-CD9/anti-CD63/anti-CD81-APC) used in the setting of the gating areas and identification of fluorescent events. X-axis: fluorescence intensity of CFSE, detected in channel 2 (Ch02). Y-axis: fluorescent intensity of the antitetraspanin mixture detected in channel 5 (Ch05). c) Background analysis of fluorescent events (left: CFSE, right: anti-tetraspanin mixture) for unstained fPBS (Buffer Control), 5 unstained PPP, 1 single-stained fPBS and 5 single-stained PPP. Black dots: individual PPP samples.
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