An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 35 IFCM gating strategy for the detection of single particles ≤400 nm in plasma EVs represent a heterogeneous group with different cellular origin. The analysis of single EVs, as well as the different subsets, will provide a better understanding of the pathophysiological state of the individual. Therefore, we designed a gating strategy to analyze individual sub-micron sized particles based on 1) the analysis of events within a pre-defined sub-micron size range, and 2) exclusion of multi-spot fluorescent events from our analysis. Based on the previous results, we selected all events with SSC intensities ≤ 900 a.u. - corresponding with particles of 400 nm and below. (Figure 2a – I). Next, we checked for multiplet detection within each separate fluorescent detection channel based on the number of fluorescent spots within the pixel grid for each acquired event: these spots were quantified by combining the “Spot Count” feature with the intensity masks for each of the channels used per experiment. Although the camera can spatially resolve signals originating from multiple simultaneously imaged EVs, the software anticipates that the signals are originating from multiple locations within 1 cell. By selecting all events that showed 0 or 1 spot, representing either negative or single-positive events for a fluorescent marker, we were able to exclude multiplet events from our analysis (Figure 2a – II, III). As a last step, we calculated the distance between individual fluorescent spots detected in different fluorescent channels to exclude any false double-positive events (defined as 2 different single-positive particles within the same event). To this end, we created a new mask by combining the intensity masks of the channels in use per experiment using Boolean logic (e.g., MC_Ch02 OR MC_Ch05), and combined this new mask with the “Min Spot Distance” feature to calculate the distance between the fluorescent spots across the detection channels used. We then excluded all fluorescent events that did not occupy the same location on the pixel grid (Figure 2a – IV). Ultimately, this gating strategy allows for the identification and subsequent analysis of single fluorescent sub-micron sized particles ≤400 nm in PPP and is applied throughout the rest of this work. Establishment of IFCM background fluorescence Given their physical characteristics, EVs yield faint fluorescent signals – compared too cells – when measured with IFCM. Therefore, we assessed the fluorescent background levels induced by our staining protocol. As no washing steps are performed, the discrimination of EVs from fluorescent background signals is
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