Chapter 4 86 Endowed with increased fluorescence detection sensitivity over conventional FCM,21 and the capability of distinguishing particles based on high-resolution imaging,22 imaging FCM (IFCM) allows quantification and characterization of uEVs to solve the mentioned difficulties and bypass EV isolation. IFCM has been demonstrated for single EV measurement in minimally processed plasma23, cell supernatant,22 and isolated uEVs,24,25 but no methodology to detect uEVs from urine without relying on prior EV purification. The current study aims to provide a protocol to characterize uEVs by IFCM directly in stained urine by excluding A-F particles and diminishing the influence of THP. RESULTS Outline of the manuscript The workflow of this study is schematically summarized in Figure 1. We aimed for standardized uEV-IFCM measurements independent of uEV purification for clinical usage. To this end, supporting techniques (TEM, NTA, TR-FIA) were used to indicate the size distribution, concentration, and uEV markers in unprocessed urine. The IFCM instrument was calibrated with standardized reference material for crossplatform comparisons and then used to quantify, phenotype and characterize uEVs in the minimally processed urine (unprocessed urine with labeling). Urine samples from healthy controls (HC) were initially used to establish the uEV-IFCM protocol, which was further developed using the urine of kidney transplant recipients (KTR).
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