TY - JOUR
T1 - Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry
AU - Wu, Liang
AU - Woud, Wouter W.
AU - Baan, Carla C.
AU - Hesselink, Dennis A.
AU - van der Pol, Edwin
AU - Jenster, Guido
AU - Boer, Karin
N1 - Funding Information: 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. Funding Information: 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
PY - 2023/2/1
Y1 - 2023/2/1
N2 - 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.
AB - 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.
KW - Extracellular vesicles
KW - Human urine
KW - Imaging flow cytometry
KW - Isolation-free methodology
KW - Kidney transplantation
UR - http://www.scopus.com/inward/record.url?scp=85144510449&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.nano.2022.102638
DO - https://doi.org/10.1016/j.nano.2022.102638
M3 - Article
C2 - 36549551
SN - 1549-9634
VL - 48
JO - Nanomedicine: Nanotechnology, Biology and Medicine
JF - Nanomedicine: Nanotechnology, Biology and Medicine
M1 - 102638
ER -