Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 89 with the bright model, the intensity of included pixels must be higher than the background.28 Therefore, the Peak Mask with a spot-to-cell background ratio of 1 was the most appropriate for identifying singlets and excluding events with >1 spot in fluorescence-detecting channels (Ch02 & Ch05). The next step, coincidence/false “singlets” (one green and one red object, but their positions did not overlap), were excluded based on the distance between Ch02 and Ch05 spots ≠ 0. Distinguishing uEV-singlets from auto-fluorescent particles in labelled urine In fDPBS, no positive particles (Figure 3A) were observed. Conversely, unprocessed urine (without staining) contained 1.6 ± 0.7 × 106 objects/mL positive particles (Figure 3B). These events are auto-fluorescent (A-F) particles and demonstrated positive fluorescent signals in fluorescence-detecting channels: Ch02, Ch03, and Ch05. Ch02 and Ch05 were used to detect Alexa488, and APC signals, respectively. No fluorescent reagent was used for Ch03, so it only presented the A-F signals. In the minimally processed urine (urine only with labeling), the distribution of A-F particles overlapped with positive uEVs (Figure 3C), necessitating the exclusion/ removal of A-F particles. Lowering laser power or detergent treatment did not sufficiently remove A-F particles (Figure 3D). Hence, these A-F particles are not phospholipid bilayer structures, and their A-F signal is unrelated to the high laser power. A step of short-run centrifugation (10,000 g × 10 min) was found to remove 98.9 ± 0.3% of A-F particles (p = 0.0058; Figure 3D). Though centrifugation effectively removed A-F particles, it could cause a loss of uEVs. We designed a gating strategy distinguishing non-A-F particles (uEVs) from A-F particles based on the absence of A-F particles in centrifuged urine and aimed to bypass centrifugation finally. First, urine was“cleane” using that centrifugation (Figure 3B to 3E). Next, centrifuged urine was stained with CD63-Alexa488 and CD63-APC to show positive uEVs (Figure 3F). The spillover from Ch02 to Ch03 was compensated (value: 0.19 in the compensation matrix), so uEVs were horizontally distributed in Figure 3G. Notably, applying this compensation to A-F particles in the unprocessed urine did not alter the A-F signal (Supplementary Figure S4). Based on the remaining events after this centrifugation, a gate,“Non-A-F Particle”, was set in Ch03 with a cutoff value of 150 A-F intensity (arbitrary unit), which is equal to 5 MESF-PE (blue gate in Figure 3G). Then, the obtained compensation matrix and gate were applied to urine samples without centrifugation (blue gate in Figure 3H). By doing so, A-F particles were excluded from the following analysis without requiring centrifugation, and therefore, urine was kept minimally processed until uEV labeling, thus bypassing potential uEV loss. The“Non-A-F Particle” gate was
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