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Article

Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry

1
Department of Medicine and Aging Sciences, University “G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
2
Center for Advanced Studies and Technology (CAST), University “G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
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Department of Pharmacy, University “G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
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Department of Medical, Oral and Biotechnological Sciences, University “G. d’Annunzio”, Chieti-Pescara, 66100 Chieti, Italy
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Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 Urbino, Italy
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Department of Experimental Medicine, “Sapienza”, University of Rome, 00161 Rome, Italy
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(21), 7885; https://doi.org/10.3390/ijms21217885
Received: 16 September 2020 / Revised: 16 October 2020 / Accepted: 21 October 2020 / Published: 23 October 2020
Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the “tip of the iceberg” of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV < 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained. View Full-Text
Keywords: extracellular vesicles (EVs); flow cytometry; standardization; Rosetta bead system extracellular vesicles (EVs); flow cytometry; standardization; Rosetta bead system
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MDPI and ACS Style

Simeone, P.; Celia, C.; Bologna, G.; Ercolino, E.; Pierdomenico, L.; Cilurzo, F.; Grande, R.; Diomede, F.; Vespa, S.; Canonico, B.; Guescini, M.; Stocchi, V.; Lotti, L.V.; Guagnano, M.T.; Stellin, L.; Papa, S.; Trubiani, O.; Marchisio, M.; Miscia, S.; Lanuti, P. Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry. Int. J. Mol. Sci. 2020, 21, 7885. https://doi.org/10.3390/ijms21217885

AMA Style

Simeone P, Celia C, Bologna G, Ercolino E, Pierdomenico L, Cilurzo F, Grande R, Diomede F, Vespa S, Canonico B, Guescini M, Stocchi V, Lotti LV, Guagnano MT, Stellin L, Papa S, Trubiani O, Marchisio M, Miscia S, Lanuti P. Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry. International Journal of Molecular Sciences. 2020; 21(21):7885. https://doi.org/10.3390/ijms21217885

Chicago/Turabian Style

Simeone, Pasquale, Christian Celia, Giuseppina Bologna, Eva Ercolino, Laura Pierdomenico, Felisa Cilurzo, Rossella Grande, Francesca Diomede, Simone Vespa, Barbara Canonico, Michele Guescini, Vilberto Stocchi, Lavinia V. Lotti, Maria T. Guagnano, Luisa Stellin, Stefano Papa, Oriana Trubiani, Marco Marchisio, Sebastiano Miscia, and Paola Lanuti. 2020. "Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry" International Journal of Molecular Sciences 21, no. 21: 7885. https://doi.org/10.3390/ijms21217885

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