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

J 2015

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

POSPÍCHALOVÁ, Vendula, Jan SVOBODA, Zankruti DAVE, Anna KOTRBOVÁ, Karol KAISER et. al.

Basic information

Original name

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

Authors

POSPÍCHALOVÁ, Vendula (203 Czech Republic, belonging to the institution), Jan SVOBODA (203 Czech Republic), Zankruti DAVE (356 India, belonging to the institution), Anna KOTRBOVÁ (203 Czech Republic, belonging to the institution), Karol KAISER (703 Slovakia, belonging to the institution), Dobromila KLEMOVÁ (203 Czech Republic, belonging to the institution), Ladislav ILKOVICS (203 Czech Republic, belonging to the institution), Aleš HAMPL (203 Czech Republic, belonging to the institution), Igor CRHA (203 Czech Republic), Eva JANDÁKOVÁ (203 Czech Republic, belonging to the institution), Luboš MINÁŘ (203 Czech Republic), Vít WEINBERGER (203 Czech Republic) and Vítězslav BRYJA (203 Czech Republic, belonging to the institution)

Edition

Journal of Extracellular Vesicles, Järfälla, Co-Action Publishing, 2015, 2001-3078

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

Genetics and molecular biology

Country of publisher

Sweden

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

RIV identification code

RIV/00216224:14310/15:00080746

Organization unit

Faculty of Science

DOI

http://dx.doi.org/10.3402/jev.v4.25530

UT WoS

000365076000001

Keywords in English

CFSE; ascites; exosomes; extracellular vesicles; flow cytometry; fluorescent labeling; lipophilic styryl dye; microvesicles; quantification

Abstract

V originále

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. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.

Links

EE2.3.30.0009, research and development project

Name: Zaměstnáním čerstvých absolventů doktorského studia k vědecké excelenci

GAP301/11/0747, research and development project

Name: Molekulární mechanismy nekanonické Wnt signalizace v leukémii

Investor: Czech Science Foundation

MUNI/M/1050/2013, interní kód MU

Name: Automatizovaná analýza elektronmikroskopických snímků pro použití v biologii a medicíně (Acronym: Analýza TEM snímků)

Investor: Masaryk University, INTERDISCIPLINARY - Interdisciplinary research projects

Citovat

POSPÍCHALOVÁ, Vendula, Jan SVOBODA, Zankruti DAVE, Anna KOTRBOVÁ, Karol KAISER, Dobromila KLEMOVÁ, Ladislav ILKOVICS, Aleš HAMPL, Igor CRHA, Eva JANDÁKOVÁ, Luboš MINÁŘ, Vít WEINBERGER and Vítězslav BRYJA. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. Journal of Extracellular Vesicles. Järfälla: Co-Action Publishing, 2015, vol. 4, No 25530, p. 1-15. ISSN 2001-3078. Available from: https://dx.doi.org/10.3402/jev.v4.25530.

@article{1232996,
   author = {Pospíchalová, Vendula and Svoboda, Jan and Dave, Zankruti and Kotrbová, Anna and Kaiser, Karol and Klemová, Dobromila and Ilkovics, Ladislav and Hampl, Aleš and Crha, Igor and Jandáková, Eva and Minář, Luboš and Weinberger, Vít and Bryja, Vítězslav},
   article_location = {Järfälla},
   article_number = {25530},
   doi = {http://dx.doi.org/10.3402/jev.v4.25530},
   keywords = {CFSE; ascites; exosomes; extracellular vesicles; flow cytometry; fluorescent labeling; lipophilic styryl dye; microvesicles; quantification},
   language = {eng},
   issn = {2001-3078},
   journal = {Journal of Extracellular Vesicles},
   title = {Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer},
   url = {http://www.journalofextracellularvesicles.net/index.php/jev/article/view/25530},
   volume = {4},
   year = {2015}
}

TY  - JOUR
ID  - 1232996
AU  - Pospíchalová, Vendula - Svoboda, Jan - Dave, Zankruti - Kotrbová, Anna - Kaiser, Karol - Klemová, Dobromila - Ilkovics, Ladislav - Hampl, Aleš - Crha, Igor - Jandáková, Eva - Minář, Luboš - Weinberger, Vít - Bryja, Vítězslav
PY  - 2015
TI  - Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer
JF  - Journal of Extracellular Vesicles
VL  - 4
IS  - 25530
SP  - 1-15
EP  - 1-15
PB  - Co-Action Publishing
SN  - 20013078
KW  - CFSE
KW  - ascites
KW  - exosomes
KW  - extracellular vesicles
KW  - flow cytometry
KW  - fluorescent labeling
KW  - lipophilic styryl dye
KW  - microvesicles
KW  - quantification
UR  - http://www.journalofextracellularvesicles.net/index.php/jev/article/view/25530
N2  - 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. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.
ER  -

POSPÍCHALOVÁ, Vendula, Jan SVOBODA, Zankruti DAVE, Anna KOTRBOVÁ, Karol KAISER, Dobromila KLEMOVÁ, Ladislav ILKOVICS, Aleš HAMPL, Igor CRHA, Eva JANDÁKOVÁ, Luboš MINÁŘ, Vít WEINBERGER and Vítězslav BRYJA. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. \textit{Journal of Extracellular Vesicles}. Järfälla: Co-Action Publishing, 2015, vol.~4, No~25530, p.~1-15. ISSN~2001-3078. Available from: https://dx.doi.org/10.3402/jev.v4.25530.

Detailed Information on Publication Record