Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 95 Figure 6 - Verification of uEV singlets in the serially diluted urine. (A) Serial dilutions were performed on the double-stained healthy urine samples (n = 5), showing the linear regression on the mean values of CD63+ uEV concentration. R2: coefficient of regression. (B, C) Dilution effects on the MESF-Alexa488 and MESF-APC of CD63+ uEV. “ns”: no significance. Application and development of the isolation-free protocol for patient’s uEV Our protocol has been developed using HC urine samples and showed good inter- and intra-reproducibility in long-term and repeated measurements for clinical applications (CV < 6.1%; Supplementary Figure S7). However, patient’s urine differs from healthy conditions, including higher pH and increased urinary protein levels (Supplementary Table S1). We found that normalizing urinary pH by the commonly used dilution with fDPBS did not alter the detection of uEV numbers (Supplementary Figure S8). Tamm-Horsfall protein (THP) is the most abundant urinary protein, likely polymerizing and entrapping uEVs, and THP level rises significantly during kidney dysfunctions.15,16,34 Using electron microscopy, most uEVs observed in healthy urine were identified as single/free structures (Supplementary Figure S2A & S2B). However, in KTR urine, many uEVs are enclosed in aggregate- or filament-like structures (Figure 7A, largearea pictures in Supplementary Figure S2C & S2D). These aggregates/filaments might be associated with higher urinary total protein in KTR urine compared with HC urine (p = 0.0050; Figure 7B). Using ELISA, KTR urine also showed significantly elevated urinary THP compared with HC urine (p = 0.0197; Figure 7C).
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