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Article

Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples

1
Department of Medicine and Aging Sciences, University “G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
2
Center for Advanced Studies and Technology (C.A.S.T.), University “G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
3
Department of Innovative Technologies in Medicine & Dentistry, University G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
4
Department of Pharmacy, University “G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
5
Department of Research and Diagnostics, IRCCS Giannina Gaslini, 16147 Genova, Italy
6
Laboratory of Clinical Proteomics, “Giovanni Paolo II” Hospital, 73100 ASL-Lecce, Italy
7
Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy
8
Department of Biomorphological Science, Molecular Genetic Institute, Italian National Research Council, 66100 Chieti, Italy
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2021, 22(1), 48; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010048
Received: 15 November 2020 / Revised: 17 December 2020 / Accepted: 19 December 2020 / Published: 23 December 2020
(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy 2.0)
Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance. View Full-Text
Keywords: extracellular vesicles; polychromatic flow cytometry; biomarkers; proteomics; fresh peripheral blood extracellular vesicles; polychromatic flow cytometry; biomarkers; proteomics; fresh peripheral blood
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MDPI and ACS Style

Marchisio, M.; Simeone, P.; Bologna, G.; Ercolino, E.; Pierdomenico, L.; Pieragostino, D.; Ventrella, A.; Antonini, F.; Del Zotto, G.; Vergara, D.; Celia, C.; Di Marzio, L.; Del Boccio, P.; Fontana, A.; Bosco, D.; Miscia, S.; Lanuti, P. Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples. Int. J. Mol. Sci. 2021, 22, 48. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010048

AMA Style

Marchisio M, Simeone P, Bologna G, Ercolino E, Pierdomenico L, Pieragostino D, Ventrella A, Antonini F, Del Zotto G, Vergara D, Celia C, Di Marzio L, Del Boccio P, Fontana A, Bosco D, Miscia S, Lanuti P. Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples. International Journal of Molecular Sciences. 2021; 22(1):48. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010048

Chicago/Turabian Style

Marchisio, Marco, Pasquale Simeone, Giuseppina Bologna, Eva Ercolino, Laura Pierdomenico, Damiana Pieragostino, Alessia Ventrella, Francesca Antonini, Genny Del Zotto, Daniele Vergara, Christian Celia, Luisa Di Marzio, Piero Del Boccio, Antonella Fontana, Domenico Bosco, Sebastiano Miscia, and Paola Lanuti. 2021. "Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples" International Journal of Molecular Sciences 22, no. 1: 48. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010048

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