Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles.
Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications.


BACKGROUND
The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology.
Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Esther N M
EN
Nolte-'t Hoen
Els J
EJ
van der Vlist
Marian
M
Aalberts
Hendrik C H
HC
Mertens
Berend Jan
BJ
Bosch
Willem
W
Bartelink
Enrico
E
Mastrobattista
Ethlinn V B
EV
van Gaal
Willem
W
Stoorvogel
Ger J A
GJ
Arkesteijn
Marca H M
MH
Wauben
D000818:Animals; D055252:Cell-Derived Microparticles / Q000648:ultrastructure*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000648:ultrastructure; D011992:Endosomes / Q000648:ultrastructure*; D055354:Exosomes / Q000648:ultrastructure*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008081:Liposomes / Q000032:analysis / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D053758:Nanoparticles / Q000032:analysis / Q000648:ultrastructure*; D012661:Semen / Q000166:cytology; D014771:Virion / Q000648:ultrastructure
D008081:Liposomes