Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 101 MATERIALS & METHODS Urine samples were measured by (1) transmission electron microscopy (TEM), (2) nanoparticle tracking analysis (NTA), (3) time-resolved fluorescence immunoassay (TR-FIA), and (4) IFCM without using any uEV isolation method. Supernatant harvested from COLO-205 cells was used as a positive control to investigate the inter- and intra-assay reproducibility during IFCM measurement. TEM, NTA, and TR-FIA protocols were introduced in supplementary materials, including the COLO-205 cell culturing, the measurement of urine protein, creatinine, pH, TammHorsfall protein, and urine treatment with dithiothreitol (DTT). Collection of urine samples Urine was collected from 5 healthy controls (HC; Medical Ethical Review number 2018-1623) and 5 kidney transplant recipients (KTR) with acute kidney injury in the first 2 weeks post-transplantation (Medical Ethical Review number 2018-035), approved by the institutional review board of the Erasmus MC. Details regarding the collection of urine samples were demonstrated in the Supplementary material. The gender, age, pH, urinary total protein concentration, and urine creatinine concentration of included individuals are shown in Supplementary Table S1. Labeling EVs from urine for IFCM CD9 and CD63 are tetraspanins playing critical roles in EV generation and excretion.26 Both are used as general uEV surface markers.27 Urine and COLO-205 cell medium (positive control) were stained with antibodies (all from Biolegend, USA): CD63Alexa488 (clone H5C6; fluorophore-to-protein ratio 5.20); CD63-APC (clone H5C6; fluorophore-to-protein ratio 1.22); IgG1-Alexa488 or IgG1-APC (both clone MOPC21). Our study aims to investigate diverse uEV populations by combining markers conjugated with different fluorophores, so two CD63 antibodies were used to demonstrate the influence of fluorophore conjugation on the uEV-IFCM detection and compare the single- and double-positive backgrounds. Antibodies (100–200 µL) were centrifuged at 16,000 g at 4 ºC for 10 min (FrescoTM 17 Microcentrifuge, Thermofisher Scientific), and only the supernatant was pipetted to avoid aggregates. Based on titration, the final concentration of all antibodies in the sample solution was 0.44 µg/mL. 112 µL of samples were incubated with 4 µL of 15-fold-diluted CD63-Alexa488 and 4 µL of 15-fold-diluted CD63-APC at 4 ºC in the
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