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Updated: Aug 16, 2025
Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry
Published on: March 17, 2015
Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry
Liang Wu1, Wouter W Woud2, Carla C Baan2
1Erasmus MC Transplant Institute, Department of Internal Medicine, University Medical Center Rotterdam Erasmus MC, 3015GD Rotterdam, the Netherlands; Department of Nephrology, The First Affiliated Hospital of Shaoyang University, 422000 Shaoyang, Hunan, China.
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.
