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- Title
Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples.
- Authors
Marchisio, Marco; Simeone, Pasquale; Bologna, Giuseppina; Ercolino, Eva; Pierdomenico, Laura; Pieragostino, Damiana; Ventrella, Alessia; Antonini, Francesca; Del Zotto, Genny; Vergara, Daniele; Celia, Christian; Di Marzio, Luisa; Del Boccio, Piero; Fontana, Antonella; Bosco, Domenico; Miscia, Sebastiano; Lanuti, Paola
- 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.
- Subjects
FLOW cytometry; BLOOD sampling; BASIC dyes; EXTRACELLULAR vesicles; PROTEOMICS; BIOMARKERS; BODY fluids
- Publication
International Journal of Molecular Sciences, 2021, Vol 22, Issue 1, p48
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms22010048