An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 49 IFCM facilitates specific EV subset analysis in contaminated/diluted PPP samples To demonstrate the discriminative capabilities of our methodology, and to show that our staining procedure is specific, we mixed human and mouse PPP at various ratios (10% increments) and stained these samples with CFDA-SE and both antihuman CD31-BV421 and anti-mouse CD31-APC mAbs. For the analysis, all CFSEpositive events <400 nm were selected, and human and mouse single EVs were identified based on the species-specific antibody, thus ensuring the analysis of double-positive events. Quantification of total human and mouse single EV in 100% human or mouse PPP revealed a ~13-fold higher concentration in human: 2.29E7 ± 6.25E6 (CFSE+ antihuman CD31+, Figure 8a) vs 1.8E6 ± 3.46E5 (CFSE+ anti-mouse CD31+, Figure 8b) objects/mL, respectively. As expected, human EV concentrations showed a linear increase as the fraction of human PPP increased (R2 = 0.95), while mouse EV showed the opposite trend (linear decreased as the fraction of human PPP increased – R2 0.81). Anti-human and anti-mouse concentrations obtained after staining the 100% human and mouse samples with their corresponding isotype controls were used to establish the background concentrations of our protocol (as indicated by the dashed red lines in Figures 8a, b), and showed that the detection of anti-human/ mouse EV is specific and above background. Additionally, no mAb cross-reactivity between species was observed. Together, these data show that our method enables the discrimination and accurate quantification of distinct single EV populations in unprocessed, mixed PPP samples.
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