Chapter 3 50 Figure 8 – Quantification of single EVs in mixed human and mouse PPP samples. Samples were stained with CFDA-SE and anti-human and anti-mouse CD31 (conjugated to BV421 and APC, respectively). Quantification of a) CFSE+ anti-human CD31+ single EV ≤400 nm showed a linear increase corresponding to the increase in human PPP abundance (R2 = 0.95), while b) CFSE+ anti-mouse CD31+ single EV ≤400 nm showed a linear decrease corresponding to the decrease in mouse PPP abundance (R2 = 0.81). Data were obtained through three independent experiments using the same human and mouse PPP samples. X-axis: v/v ratio of mouse – human PPP. Data shown represent the mean ± standard error. Red dashed lines: background concentrations of our protocol as indicated by the measurement of isotype controls. DISCUSSION We developed an IFCM-based methodology to identify, phenotype and determine the concentration of single EVs from molecular complex blood plasma without prior isolation, providing an advantage over currently available analytical techniques, which do require EV isolation. We present an easy-to-use sample processing and staining protocol (Figure 9), and provide a gating strategy for the identification of single EVs. Following this gating strategy, EV subpopulations in PPP could be readily discerned based on the colocalization of two fluorescent markers bound to EV membranes. Additionally, platform standardization through both size and fluorescence calibration allows reproducibility and comparison of acquired data, showing the potential of our method for translation into clinical application. Given that neither the isolation of EVs from PPP nor sample washing after staining with fluorescently labelled mAbs was performed, it was imperative to assess the fluorescent background levels induced by our sample handling protocol. Using control samples, we showed minimal background fluorescence and clear discrimination of specific fluorescent events above background. Approaches taken by other groups analyzing EV in PPP using IFCM involve sample isolation 30 and/ or washing steps to remove unbound mAbs 26,30; here we show that such sample isolation and/or washing steps can be omitted by detecting and eliminating the
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