An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 47 Figure 7a shows the ERF calibrated (APC calibration performed as described in Figure 4, for BV421 calibration see Supplementary Figure 1) IFCM results after double staining of both fPBS and a representative PPP sample with anti-CD9-APC and anti-CD31-BV421 and subsequent detergent treatment. The lower fluorescent threshold for Ch01 (BV421) was established at 677.71 ERF; upper fluorescent threshold was established at 112,201 ERF. A visual representation of the events before detergent treatment within each gate is shown in Figure 7b. As stated before, only single spot fluorescent events (with colocalized fluorescent spots for double-positive events) were analyzed. Focusing on double-positive particles, we acquired a total of 5.12E7 ± 1.02E7 objects/ mL before detergent treatment and 3.61E6 ± 5.46E6 objects/mL (~7% of total events) after detergent treatment, thus showing that ~93% of the double-positive events detected in the PPP sample could be classified as true single EVs with this strategy. Mean ERF values of the double-positive events in all 5 PPP samples (before detergent treatment) were calculated at ~7,620 (range 3,640 – 9,240) and 20.4 (range 15 – 27.9) for BV421 and APC, respectively. Additionally, analysis of fPBS + mAbs (both anti-CD9 and anti-CD31 antibodies), PPP + isotype controls and fPBS + isotope controls yielded particle concentrations within the previously established fluorescent background range (~E5 objects/mL), both before and after detergent treatment - indicating that the double-positive single EV detected in the PPP + mAb samples were detected well above the level of the background concentrations (Figure 7c). Thus, the staining of PPP samples with anti-CD9 and anti-CD31 showed that double-positive events (before detergent treatment) can be successfully identified as true single EV. Although this staining approach (the combination of two surface markers expressed on EV) differs from the previously used staining approach (the combination of CFDA-SE and the anti-tetraspanin antibody mixture), both strategies resulted in the identification of true EV on the basis of the colocalization of two fluorophores within the same event – indicating that this colocalization is membrane facilitated and therefore can be used as a criteria to identify EV in unprocessed PPP.
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