Validation of EV sample quality. A. Electron microscopy (EM) shows typical round morphology and size range of urinary EVs. B. Urinary EVs with multiple membrane layers (arrows) were seen occasionally. C.-D. Immuno-labelling of EV-markers CD59 and CD63 showed their presence on the urinary EVs by EM. E. The size distribution of the urinary EVs in the EM images indicated that most of the EVs were small, < 100 nm in diameter (N = 3, total of 425 EVs). F . Platelet EVs showed typical morphology and somewhat larger size than urinary EVs by EM. G. Western blotting of the samples from each step of the urinary EV isolation confirmed the enrichment of CD9, TSG101, CD59 and CD63 as well as the absence of cellular organelle proteins calnexin, TOMM20 and GM130 in the EVs. Equal quantities of protein were loaded from all urine-derived samples. Supernatant (sn).
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