| Literature DB >> 33374539 |
Marco Marchisio1,2, Pasquale Simeone1,2, Giuseppina Bologna1,2, Eva Ercolino1,2, Laura Pierdomenico1,2, Damiana Pieragostino2,3, Alessia Ventrella4, Francesca Antonini5, Genny Del Zotto5, Daniele Vergara6,7, Christian Celia4, Luisa Di Marzio4, Piero Del Boccio2,4, Antonella Fontana4, Domenico Bosco8, Sebastiano Miscia1,2, Paola Lanuti1,2.
Abstract
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.Entities:
Keywords: biomarkers; extracellular vesicles; fresh peripheral blood; polychromatic flow cytometry; proteomics
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Year: 2020 PMID： 33374539 PMCID： PMC7793062 DOI： 10.3390/ijms22010048
Source DB: PubMed Journal: Int J Mol Sci ISSN： 1422-0067 Impact factor: 5.923