Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 93 Background analysis Without performing any wash steps, it was essential to verify the presence of CD63+ uEVs. First, detergent treatment was used to check if the readouts of IFCM represent biological membrane structures. Before detergent treatment, for all HC urine samples (double-CD63-stained), we found that the majority of readouts were“+” particles, 3.8 ± 1.3 × 107 objects/mL (Figure 5A). After detergent treatment,“+” events decreased to 4.8 ± 2.7 × 105 objects/mL, representing a 98.5 ± 0.9% decrease compared to no detergent (p = 0.0034). As for the single-positive particles in the stained urine, 3.2 ± 2.5 × 106 objects/mL of events were“CD63-Alexa488” (Figure 5B), and 5.8 ± 0.6 × 105 objects/mL of events were“CD63-APC” (Figure 5C). After detergent treatment, the concentration of the single“CD63-Alexa488” events and the“CD63-APC” events was reduced by 91.3 ± 6.7% and 77.9 ± 30.1%, respectively (Figures 5B & 5C). Next to detergent treatment, the double-positive concentrations in other controls were summarized in Figure 5A. Compared with unstained, isotype-stained, and double-stained plus detergent-treated urine, the average uEV-to-background concentration ratio for the double-positive region in double-stained urine is 3102.2fold, indicating a convincing presence of CD63+ uEVs in the minimally processed urine. Compared to urine with other treatments, the average uEV-to-background ratios of Alexa488 single-positive and APC single-positive events in the doublestained urine were 10.9-fold and 26.6-fold, respectively (Figures 5B & 5C). Double-positive particles are the majority of CD63+ uEVs and showed the highest uEV-to-background ratio, so they were selected to represent CD63+ uEVs in the subsequent analysis.
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