Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 87 Figure 1 - Schematic workflow of this study. Measuring uEVs by TEM, NTA, and TR-FIA In HC urine samples imaged by TEM, uEV-like cup-shaped structures were observed with diameters between 50 and 100 nm (Figure 2A, large-area images in Supplementary Figure S2). With NTA, we summarized the single-particlesize reports from all HC urine samples. We found that 93-98 % of urine particles’ diameter was <400 nm, but 2-7 % of all detected particles showed a size from 400 nm to 1200 nm (Figure 2B). The total concentration of particles measured by NTA was around 108/mL, which is an order of magnitude estimate of particles exceeding the lower limit of detection (LoD).31 We estimate the LoD to be ~90 nm based on the distribution mode. To select appropriate labeling for uEV detection by IFCM, we compared the relative expression levels of the tetraspanins CD9 and CD63 on uEVs using TR-FIA. In Figure 2C, CD63 was 6.7-fold higher than CD9 following the Europium intensity (p = 0.0302), corresponding with previous research.32 Hence, CD63+ uEV was chosen as a targeted population to demonstrate the following isolation-free uEV-IFCM methodology.
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