Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry

Liang Wu*, Wouter W. Woud, Carla C. Baan, Dennis A. Hesselink, Edwin van der Pol, Guido Jenster, Karin Boer

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.

Original languageEnglish
Article number102638
JournalNanomedicine: Nanotechnology, Biology, and Medicine
Volume48
DOIs
Publication statusPublished - Feb 2023

Bibliographical note

Acknowledgments
We thank the support from the China Scholarship Council (No. 202008430154) and Wenda Verschoor and Rens Kraaijeveld
for helping solve the technical questions of IFCM and structure this paper. We acknowledge Natasja Dits at the Department of
Urology (Erasmus Medical Center; EMC) for the TEM images and Manou van Alphen for the support of TR-FIA. All figures
were created with BioRender.com.

Publisher Copyright: © 2022 The Authors

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