Detail
Article
Online article
FT
Medvik - BMC
  • Something wrong with this record ?

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

V. Pospichalova, J. Svoboda, Z. Dave, A. Kotrbova, K. Kaiser, D. Klemova, L. Ilkovics, A. Hampl, I. Crha, E. Jandakova, L. Minar, V. Weinberger, V. Bryja,

. 2015 ; 4 (-) : 25530.

Language English Country Sweden

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc15023757

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: ~80-200 nm, microvesicles: ~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.

References provided by Crossref.org

000      
00000naa a2200000 a 4500
001      
bmc15023757
003      
CZ-PrNML
005      
20210204123608.0
007      
ta
008      
150709s2015 sw f 000 0|eng||
009      
AR
024    7_
$a 10.3402/jev.v4.25530 $2 doi
035    __
$a (PubMed)25833224
040    __
$a ABA008 $b cze $d ABA008 $e AACR2
041    0_
$a eng
044    __
$a sw
100    1_
$a Pospichalova, Vendula $u Faculty of Science, Masaryk University, Brno, Czech Republic.
245    10
$a Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer / $c V. Pospichalova, J. Svoboda, Z. Dave, A. Kotrbova, K. Kaiser, D. Klemova, L. Ilkovics, A. Hampl, I. Crha, E. Jandakova, L. Minar, V. Weinberger, V. Bryja,
520    9_
$a 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: ~80-200 nm, microvesicles: ~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.
655    _2
$a časopisecké články $7 D016428
700    1_
$a Svoboda, Jan $u Cytometry and Microscopy Facility, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Bio-port Europe, Svinare, Czech Republic.
700    1_
$a Dave, Zankruti $u Faculty of Science, Masaryk University, Brno, Czech Republic.
700    1_
$a Kotrbova, Anna $u Faculty of Science, Masaryk University, Brno, Czech Republic.
700    1_
$a Kaiser, Karol $u Faculty of Science, Masaryk University, Brno, Czech Republic.
700    1_
$a Klemova, Dobromila $u Faculty of Medicine, Masaryk University, Brno, Czech Republic.
700    1_
$a Ilkovics, Ladislav $u Faculty of Medicine, Masaryk University, Brno, Czech Republic.
700    1_
$a Hampl, Ales $u Faculty of Medicine, Masaryk University, Brno, Czech Republic.
700    1_
$a Crha, Igor $u Department of Obstetrics and Gynecology, Faculty Hospital Brno, Brno, Czech Republic.
700    1_
$a Jandakova, Eva $u Department of Pathology, Masaryk University and University Hospital Brno, Brno, Czech Republic.
700    1_
$a Minar, Lubos $u Department of Obstetrics and Gynecology, Faculty Hospital Brno, Brno, Czech Republic.
700    1_
$a Weinberger, Vit $u Department of Obstetrics and Gynecology, Faculty Hospital Brno, Brno, Czech Republic.
700    1_
$a Bryja, Vitezslav $u Faculty of Science, Masaryk University, Brno, Czech Republic. Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic; bryja@sci.muni.cz.
773    0_
$w MED00188113 $t Journal of extracellular vesicles $x 2001-3078 $g Roč. 4, č. - (2015), s. 25530
856    41
$u https://pubmed.ncbi.nlm.nih.gov/25833224 $y Pubmed
910    __
$a ABA008 $b sig $c sign $y a $z 0
990    __
$a 20150709 $b ABA008
991    __
$a 20210204123605 $b ABA008
999    __
$a ind $b bmc $g 1084089 $s 906750
BAS    __
$a 3
BAS    __
$a PreBMC
BMC    __
$a 2015 $b 4 $c - $d 25530 $i 2001-3078 $m Journal of extracellular vesicles $n J Extracell Vesicles $x MED00188113
LZP    __
$a Pubmed-20150709

Find record

Citation metrics

Archiving options