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Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

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    0472803 - MBÚ 2017 RIV GB eng J - Článek v odborném periodiku
    Pospíchalová, V. - Svoboda, Jan - Dave, Z. - Kotrbová, A. - Kaiser, K. - Klemová, D. - Ilkovics, L. - Hampl, A. - Crha, I. - Jandáková, E. - Minář, L. - Weinberger, V. - Bryja, Vítězslav
    Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer.
    Journal of Extracellular Vesicles. Roč. 4, March 31 (2015), s. 25530. E-ISSN 2001-3078
    Institucionální podpora: RVO:61388971 ; RVO:68081707
    Klíčová slova: exosomes * microvesicles * extracellular vesicles
    Kód oboru RIV: EE - Mikrobiologie, virologie

    Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: similar to 80-200 nm, microvesicles: similar to 200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein-and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers.
    Trvalý link: http://hdl.handle.net/11104/0270026

     
     
Počet záznamů: 1  

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