BBA - Biomembranes 1860 (2018) 1350–1361

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BBA - Biomembranes
journal homepage: www.elsevier.com/locate/bbamem

PKH26 labeling of extracellular vesicles: Characterization and cellular T

internalization of contaminating PKH26 nanoparticles
Pia Pužar Dominkuša,1, Matjaž Stenovecb,c,1, Simona Sitard, Eva Lasičc, Robert Zorecb,c,
⁎
Ana Plemenitaša, Ema Žagard, Marko Kreftb,c,e, Metka Lenassia,
a
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia
b
Celica BIOMEDICAL, Tehnološki park 24, Ljubljana, Slovenia
c
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, Ljubljana, Slovenia
d
Department of Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia
e
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot, 111, Ljubljana, Slovenia

A R T I C LE I N FO A B S T R A C T

Keywords: PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the
Exosomes exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite
PKH26 wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated
Fluorescent dye fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we
Nanoparticles
have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls
Confocal microscopy
Asymmetrical-flow field-flow fractionation
stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures,
using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering
detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive
particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable
from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled
exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exo-
somes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-
labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome
recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining.
Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments
as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs
that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies,
sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of
PKH26 nanoparticles.

1. Introduction
PKH dyes, such as PKH67 and PKH26, are highly fluorescent, lipo-
philic long-chain carbocyanine dyes that are used to stain artificial and
biological membranes. The aliphatic tail of these dyes rapidly inter-
calates into the exposed lipid bilayer and forms strong noncovalent
interactions, thereby promoting long-term dye retention and stable
fluorescence [1]. In cells, the whole plasma membrane becomes stained
through lateral diffusion of these dyes, which also spread to in-
tracellular membrane organelles due to membrane turnover [2]. Cell
growth, viability, and proliferation are generally not affected, and PKH
dyes have been used extensively for labeling cells [3,4], in in vivo and
in vitro cell-tracking studies [5–7], and for visualizing homing and
proliferation of recirculating hematopoietic stem and progenitor cells
[8]. PKH dyes generally have an easy and rapid staining procedure,
with simple dye detection by flow cytometry and fluorescent

Abbreviations: AF4-MALS, asymmetrical-flow field-flow fractionation coupled to multi-angle light-scattering detector; DPBS, Dulbecco's phosphate-buffered saline; EVs, extracellular
vesicles; Rgeom, geometric radius; Rrms, root-mean-square radius
⁎
Corresponding author at: Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov Trg 2, SI-1000 Ljubljana, Slovenia.
E-mail addresses: pia.puzar-dominkus@mf.uni-lj.si (P. Pužar Dominkuš), Matjaz.Stenovec@mf.uni-lj.si (M. Stenovec), Simona.Sitar@ki.si (S. Sitar), eva.lasic@mf.uni-lj.si (E. Lasič),
Robert.Zorec@mf.uni-lj.si (R. Zorec), ana.plemenitas@mf.uni-lj.si (A. Plemenitaš), ema.zagar@ki.si (E. Žagar), marko.kreft@mf.uni-lj.si (M. Kreft),
metka.lenassi@mf.uni-lj.si (M. Lenassi).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.bbamem.2018.03.013
Received 26 July 2017; Received in revised form 14 February 2018; Accepted 13 March 2018
Available online 16 March 2018
0005-2736/ © 2018 Elsevier B.V. All rights reserved.
P. Pužar Dominkuš et al.
microscopy. However, there are some concerns that remain when using
these and other fluorescent probes, including noncell-associated signals
[2,4], dye transfer to adjacent or dead cells [9,10], dye cytotoxicity due
to cell over-labeling [11], and dye phototoxicity due to over-exposure
[12].
Recently, PKH membrane dyes have also been applied to studies of
extracellular vesicles (EVs) and their functions [13,14]. EVs are a het-
erogeneous population of membrane vesicles that are shed from cells in
vitro or are released into various body fluids in vivo [15]. They have
been detected at relevant concentrations in blood [16], cerebrospinal
fluid [17], urine [18,19], semen [20], breast milk, saliva [21], amniotic
fluid [22], bronchoalveolar lavage [23,24], and synovial fluid [24].
Numerous studies support the role of EVs in various physiological
processes, such as embryonic development [25], synaptic plasticity
[26], liver metabolism [27,28], and antigen presentation [29]. They
have also been shown to be involved in promoting pathologies through
modulation of immune responses [30], tumor invasiveness and metas-
tasis [31,32], resistance to drugs [33], and transfer of toxic proteins
[34–37]. This functional diversity of EVs is reflected in their distinctive
protein, lipid, and nucleic acid cargoes, which mirror those of the cell of
origin [38]. According to their size and site of formation, EVs can be
divided into two main groups: exosomes, which range from 30 nm to
100 nm in diameter and are formed as intraluminal vesicles in multi-
vesicular bodies, which fuse with the plasma membrane for their re-
lease; and microvesicles, which are larger structures from 100 nm to
1000 nm in diameter that are formed directly at the plasma membrane
through outward budding [39].
Functionally, EVs can interact with their target cells through one of
a number of processes, including: fusion or hemi-fusion with the plasma
membrane, followed by the “injection” of their intraluminal contents
into the cytosol; ligand-receptor binding, to modulate intracellular
signaling pathways; internalization through clathrin-dependent en-
docytosis and clathrin-independent pathways, such as caveolin-medi-
ated uptake, macropinocytosis, phagocytosis, and lipid-raft-mediated
endocytosis [40–42]. As inhibitors of specific internalization pathways
fail to completely abrogate EV uptake by cells, it appears that EVs can
be internalized via more than one route [42]. EV uptake by cells can be
visualized directly by use of fluorescent lipid membrane dyes that stain
the EV membranes. By labeling EVs with PKH26 or PKH67, the inter-
actions with and uptake of EVs by epithelial and breast cancer cells
[13], macrophages [43], dendritic cells [29], and endothelial and
myocardial cells [44] have been followed using fluorescence micro-
scopy and flow cytometry. Similarly, EV uptake by bladder cancer cells
has been studied using imaging flow cytometry [14]. PKH67 pre-la-
beled EVs isolated from dendritic cells have also been used in quanti-
tative and qualitative analyses using high-resolution flow cytometry
[45].
Despite the wide use of PKH-labeled EVs in cell targeting and
functional studies, nonEV-associated fluorescent structures have never
been systematically characterized, and their internalization by cells has
never been evaluated. The need for such studies was supported by Lai
et al. [46], who showed that staining with PKH67 resulted in control
samples (i.e., without EVs) that had greater fluorescent signals than
PKH67-stained EVs. Additionally, tissue macrophages have been shown
to actively acquire various labels in vivo [47], and endocytosis of mi-
celles has been exploited for cellular uptake of nanomedicines [48].
Thus, the nonEV-associated fluorescent structures present in PKH-la-
beled EV samples can compromise the interpretation of EV inter-
nalization and functional studies, and especially when the appropriate
EV-free controls are not included.
Here, the aim was thus to characterize the nonEV-associated fluor-
escent structures in PKH26-labeled EV samples that were stained using
procedures based on ultracentrifugation [46], filtration through Vi-
vaspin concentrators [21,49], and sucrose cushion [50] and sucrose
gradient [45] purification. The input samples were exosomes that were
isolated from culture supernatants from a lymphoblastoid B cell line,
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BBA - Biomembranes 1860 (2018) 1350–1361
and PKH26 dye resuspended in particle-free buffer as the control. The
size, number density, surface area, and fluorescence intensity of the
particles in these PHK26-stained samples were determined using con-
focal microscopy and asymmetric-flow field-flow fractionation coupled
to a multi-angle light-scattering detector (AF4-MALS). These data were
used to critically evaluate the suitability of the staining procedures.
Internalization of these PHK26-stained samples was also investigated
using live-cell confocal microscopy, to examine the relevance of the
nonEV-associated fluorescent structures in terms of EV internalization
and functional studies.
2. Materials and methods
2.1. Exosome staining
Lyophilized exosome standard that was purified from the lympho-
blastoid B cell-line culture media (HansaBioMed, Estonia) was re-
suspended in ultra-pure water to a protein concentration of 1.0 μg/μL,
following the manufacturer instructions. The resuspended exosomes
were then stained with PKH26 using PKH26 Red Fluorescent Cell Linker
Kits for General Cell Membrane Labeling (Sigma-Aldrich). Prior to
staining, PKH26 in diluent C was incubated in an ultrasonic water bath
at 37 °C for 15 min. Alternatively, for the control sample, particle-free
Dulbecco's phosphate-buffered saline (DPBS; Sigma-Aldrich) was used
as the input instead of the exosome standard. Exosome and control
samples were stained according to the following procedures.
2.1.1. Procedure A
PKH26 dye was diluted in 100 μL diluent C to a final concentration
of 8 μM (dye solution). Then 10 μg of exosomes (AF4-MALS analysis,
20 μg) in 20 μL DPBS were diluted with 80 μL diluent C, added to the
dye solution, and incubated for 5 min while mixed with gentle pipet-
ting. Excess dye was bound with 100 μL 10% exosome-depleted fetal
bovine serum (Sigma-Aldrich) in Dulbecco's modified Eagle's medium
(Sigma-Aldrich). Then the exosomes were diluted to 1 mL with DPBS
and pelleted by ultracentrifugation at 100000 ×g for 1 h 10 min at 4 °C
(TLA-55; Beckman Coulter). The pellet was gently resuspended in 50 μL
DPBS.
2.1.2. Procedure B
Exosomes were stained as described in procedure A. Then they were
diluted to 2 mL with DPBS, transferred to Vivaspin20 300-kDa MWCO
filters (Sartorius Stedim Biotech GmbH), and centrifuged at 4000 ×g
for 3 min at 4 °C. The exosomes were washed three times with 2 mL
DBPS before being concentrated to a final volume of 50 μL.
2.1.3. Procedure C
Exosomes were stained as described in procedure A. Then they were
diluted to 10 mL with DPBS and placed on top of 2 mL 20% sucrose
(Millipore) in DPBS. The exosomes were pelleted by ultracentrifugation
at 100000 ×g for 1 h 10 min at 4 °C (MLA-50; Beckman Coulter). The
pellet was gently resuspended in 50 μL DPBS.
2.1.4. Procedure D
Exosomes were stained as described in procedure A. Then they were
diluted to 400 μL with DPBS and placed onto a 20% to 60% dis-
continuous sucrose gradient. The gradient was ultracentrifuged at
100000 ×g for 18 h at 4 °C (MLS-50; Beckman Coulter). Fractions 3 to 6
(density range 1.08–1.15 g/mL as measured by refractometer, data not
shown) were combined, and fractions 7 to 10 (1.17–1.23 g/mL) were
combined, and each diluted to 30 mL with DPBS. The exosomes were
pelleted by ultracentrifugation at 100000 ×g for 1 h 10 min at 4 °C
(MLA-50; Beckman Coulter). The pellets were gently resuspended in
50 μL DPBS.
P. Pužar Dominkuš et al.
2.2. Characterization of PKH26-positive particles
2.2.1. Confocal microscopy, image analysis, and statistical analysis
Small volumes (6 μL) of gently premixed PKH26-labeled control
(i.e., particle-free DPBS) and exosome samples were transferred to
coverslips coated with 10% poly-L-lysine (to favor strong adhesion),
sealed to the objective glass, and transferred to a confocal microscope
for observation (LSM 780; Zeiss). Manual focusing was performed to
obtain the sharpest images of the bright fluorescent particles, which
were monitored within a rectangular field of 2030 μm2 by a plane-
apochromatic oil-immersion objective (63×/NA 1.4). PKH26 was ex-
cited using a 561-nm diode-pumped solid state laser line (2%), and
emission fluorescence was filtered with a 565-nm to 605-nm band-pass
filter. The settings for the detection of PKH26 emission fluorescence
were kept constant for the given sample pair of the PKH26-labeled
exosome samples and the corresponding controls (procedures A–D).
Confocal images were exported to tiff files that were analyzed using
the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Each PKH26-positive particle of several adjacent pixels with fluores-
cence intensity ≥51 gray-scale levels (intensity scale, 0–255) was ex-
tracted from the image using the 20% intensity threshold level. The
minimum size taken to identify PKH26-positive particles was three
adjacent pixels (x = 0.088 μm), and thus the minimum surface area
covered by a fluorescent particle was 0.023 μm2. The total number of
PKH26-positive particles per image, the surface area, and the fluores-
cence intensity were determined for the PKH26-labeled exosome sam-
ples and the corresponding controls (procedures A–D). All measured
parameters are reported as means ± standard error of the means
(SEM). Statistical significance was determined using Mann-Whitney U
tests, using SigmaPlot 11.0 (Systat Software Inc., USA).
2.2.2. Asymmetric-flow field-flow fractionation coupled to a multi-angle
light-scattering detector and data analysis
Asymmetric-flow field-flow fractionation separations were per-
formed on a system (Eclipse3+; Wyatt Technology Europe GmbH,
Dernbach, Germany) connected to an isocratic pump, an on-line va-
cuum degasser, and an autosampler (all 1260 series; Agilent
Technologies, USA). The samples were separated in a trapezoidal-
shaped channel with a 350-μm spacer and a Nadir Cellulose
Regenerated Cellulose membrane with a 10-kDa cut-off. The fractio-
nated particles were detected using a MALS detector (Dawn Heleos,
Wyatt Technology, USA) at 658 nm, which was calibrated using toluene
and normalized with bovine serum albumin protein as an isotropic
scatterer standard. Phosphate-buffered saline (pH 7.4) was the running
eluent, and was composed of 137 mM NaCl, 2.68 mM KCl, 10.14 mM
Na2HPO4, 1.84 mM KH2PO4, supplemented with 0.02% (w/v) sodium
azide as a bactericide, and filtered through 0.45-μm pore-size mem-
brane (Nylon 66; Supelco Analytical, USA). An additional filter with
0.1-μm pore size was placed between the HPLC pump and the AF4
channel (in a PEEK Inline Filter Holder).
Thirty-five microliters of gently premixed samples (PKH26 solution,
PKH26-labeled controls [i.e., particle-free DPBS], exosome solution,
PKH26-labeled exosome samples) were injected during a focusing/in-
jection step of 5 min, with a focus flow rate of 1.5 mL/min and an in-
jection flow rate of 0.2 mL/min, followed by a focusing/relaxation step
for an additional 7 min. The elution step was performed with two linear
cross-flow gradients, a high initial gradient of 0.275 mL/min2 (i.e.,
cross-flow from 3.0 mL/min to 0.25 mL/min in 10 min) followed by a
lower gradient of 0.0036 mL/min2 (cross-flow from 0.25 mL/min to
0.09 mL/min in 45 min). Here, 0.09 mL/min was the lowest cross-flow
limit of the instrument. Once this limit was reached, the cross-flow was
turned off. The detector flow rate was 1 mL/min. The last step was
washing the channel for 10 min in elution mode without any cross-flow.
The Astra 5.3.4.20 software (Wyatt Technology; Santa Barbara,
USA) was used to analyze the data. The size of the exosomes was ex-
pressed as two different radii; i.e., the root mean square radius (Rrms)
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BBA - Biomembranes 1860 (2018) 1350–1361
and the geometric radius (Rgeom). These radii were obtained from the
MALS detector without the need for the solute concentration and the
increment in the sample refractive index. The Rrms of the fractionated
exosomes were calculated using the data from 15 angles from the MALS
detector, applying a Debye second-order model. The Rgeom were de-
termined by the Astra Particle template assuming spherical exosome
shape. The number density of particles (particles/mL) was evaluated by
the Astra Number density template, using an exosome refractive index
of 1.39 [51]. The recovery of exosomes processed by staining proce-
dures A and C was calculated by dividing the experimental yield
(number density of unstained exosome output) by the theoretical yield
(number density of unstained exosome input) and was expressed as
percentage.
2.3. Cellular internalization of PKH26-positive particles
2.3.1. Cell culture
Primary astrocyte cultures were prepared from cortices of 3-day-old
female Wistar rats (obtained from Medical Experimental Centre at the
Institute of Pathology, University of Ljubljana, Slovenia) as described
previously [52]. The experimental animals were cared for in ac-
cordance with European and Slovenian legislation (Official Gazette of
the RS 38/13; UVHVVR, No·U34401-47/2014/7). The cells were sub-
cultured onto 22-mm-diameter poly-L-lysine-coated coverslips and used
within 4 days of plating. The cells were maintained in high glucose
Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Wal-
tham, MA, USA) containing 10% fetal bovine serum, 1 mM pyruvate,
2 mM glutamine, 5 U/mL penicillin and 5 μg/mL streptomycin, at 37 °C
in a 95% air/5% CO2 atmosphere. Unless stated otherwise, all chemi-
cals were obtained from Sigma-Aldrich (Munich, Germany) and were of
the highest purity grade available.
2.3.2. Live-cell confocal imaging
Cultured astrocytes were co-incubated with either PKH26-labeled
control (i.e., particle-free DPBS; con-procedure C) or PKH26-labeled
exosome (exo-procedure C) samples for 3 h at 37 °C, in serum-free
medium. In a subset of experiments, 20 μM fluorescent dextran (Alexa
Fluor 488, 3000 MW, D34682, Thermo Fisher Scientific) was also added
to the cells together with the samples. After labeling, the cells were
washed with extracellular solution (131.8 mM NaCl, 1.8 mM CaCl2,
2 mM MgCl2, 5 mM KCl, 10 mM D-glucose, 10 mM HEPES/NaOH,
pH 7.2), mounted into the recording chamber, supplied with extra-
cellular solution, and transferred to the microscope for observation. Z-
stacked confocal images of fluorescent compartments in live cells were
acquired as stated above. Alexa Fluor 488 was excited using a 488-nm
argon laser line (2%) and emission fluorescence was filtered with the
500-nm to 550-nm band-pass filter. The settings for the detection of the
PKH26 emission fluorescence were constant in cells loaded with
PKH26-labeled control and PKH26-labeled exosome samples.
2.3.3. Analysis of the PKH26-positive intracellular compartments after co-
incubation of primary astrocytes with PKH26-positive particles
The total number of PKH26-positive compartments per cell, the
volume and the fluorescence intensity were analyzed (3D Object
Counter plugin for ImageJ; National Institutes of Health, Bethesda, MD,
USA) in z-stacked confocal images on the basis that individual com-
partment consisted of three to 400 adjacent voxels
(0.132 × 0.132 × 0.601 μm); thus a broad span of PKH26-positive
compartments with different apparent sizes and intensities imaged at
different z positions were analyzed. All measured parameters are re-
ported as means ± SEM. Statistical significance was determined using
Mann-Whitney U tests, using SigmaPlot 11.0 (Systat Software Inc.,
USA).
P. Pužar Dominkuš et al.
Fig. 1. Schematic representation of staining procedures used to label exosomes with
PKH26 dye.
Exosomes (exo) were labeled using staining procedures based on ultracentrifugation
(procedure A), filtration through Vivaspin concentrators (procedure B), and sucrose
cushion (procedure C) and sucrose gradient (procedure D) purification. Key differences
between the procedures are highlighted by the green text. ∂, centrifugation. For details,
see Section 2.1.
3. Results
3.1. Ultracentrifugation-based exosome staining generates numerous
PKH26 nanoparticles with dimensions similar to PKH26-labeled exosomes
PKH membrane dyes are being used increasingly in studies of EVs
and their functions, although no comprehensive study of nonEV-asso-
ciated fluorescent structures present in PKH-stained EV samples has
been carried out. The existence of nonEV-associated PKH-positive
structures is supported by Lai et al. [46], who showed that staining with
PKH67 resulted in controls (PKH67-stained medium depleted of EVs)
with greater fluorescence signals than the PKH67-stained EV samples.
To characterize these nonEV-associated PKH-positive structures here,
exosomes and particle-free DPBS (control) were labeled with PKH26
following the ultracentrifugation-based staining procedure (Fig. 1,
procedure A), with the PKH26-labeled samples later analyzed using
confocal microscopy. For exosomes, exosome standards purified from
the culture supernatant of a lymphoblastoid B cell line were used
(provided by HansaBioMed), which we previously characterized for
purity, morphology, size and number density using electron
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BBA - Biomembranes 1860 (2018) 1350–1361
Fig. 2. PKH26-labeled exosome-free controls and exosome samples prepared using pro-
cedure A.
(a–d) Representative confocal images of PKH26-positive particles in a PKH26-labeled
exosome-free control (i.e., particle-free DPBS; con) (a) and in a PKH26-labeled exosome
(exo) (b) sample prepared following procedure A. Inset: Magnified selected PKH26-po-
sitive particle. Scale bars: 2 μm; inset, 0.4 μm. (c, d) Black and white image masks from
(a), (b) after image segmentation for individual PKH26-positive particles, as exosome-free
control (c; 72 particles) and exosome (d; 577 particles) samples, respectively. Scale bars:
2 μm. (e–g) Quantification of the PKH26-positive particles per image field of 2030 μm2, as
the numbers (e; N°), surface areas (f; S), and fluorescence intensities (g; Int.). Data are
means ± SEM. Numbers (bottom, e) indicate confocal images analyzed, whereas num-
bers (top, f) indicate PKH26-positive particles analyzed in f and g. ***, p < 0.001 versus
control (con; Mann-Whitney U tests).
microscopy, AF4-MALS, dynamic light scattering, and nanoparticle
tracking analysis [53]. The PKH26-positive particles in the confocal
images were analyzed for the number of particles per image, their
surface area, and their fluorescence intensity (Fig. 2).
The confocal images revealed fluorescent particles of heterogeneous
sizes and fluorescence intensities (Fig. 2a, b) in the PKH26-labeled
exosome-free controls and PKH26-labeled exosome samples prepared
using procedure A. More fluorescent particles were observed in the
exosome-containing samples than in the corresponding exosome-free
controls (Fig. 2a, b), as also indicated by the black and white mask
images obtained after segmentation of the confocal images (Fig. 2c, d).
There were 579 ± 8 (n = 20) versus 72 ± 3 (n = 20) PKH26-positive
particles per image in the exosome-containing samples and exosome-
free controls, respectively (Fig. 2e). The particle surface areas were
P. Pužar Dominkuš et al. BBA - Biomembranes 1860 (2018) 1350–1361

similar, whereas the particle fluorescence intensities were significantly Table 1

smaller in the exosome-containing samples than the exosome-free AF4-MALS measurements of the PKH26-labeled exosome-free (Control) and exosome
samples prepared using procedures A and C.
controls (Fig. 2f, g).
Of note, the measured areas of the fluorescent particles did not re- Sample Preparation Radius (nm) Number densityb
flect their actual areas, as the point-spread function contributes to some
degree of spreading (i.e., blurring) in the images of the particles. To test Procedure Rrms Rgeom (particles/mL × 1010)
this empirically, sub-diffraction fluorescent beads of known diameters a
Exosome standard 85 101 3.85
were imaged, and their diameters were calculated from the confocal Control A 77 84 0.75
images under improved contrast and assuming spherical bead geo- Exosomes A 79 94 10.70
metry. With fluorescent beads of 200-nm diameter, which is close to the Control C 65 70 0.79
theoretical lateral resolution of the fluorescent microscope Exosomes C 81 93 1.13

(d = 202.5 nm; d = λem/2NA; λem = 567 nm; NA = 1.4), the calcu- a

lated bead diameter of 206 ± 8 nm (n = 287) corresponded to the
actual diameter given. With fluorescent beads of 48-nm diameter, the
calculated bead diameter of 155 ± 3 nm (n = 413) exceeded the given
diameter by 3.2-fold. Therefore, the images of the fluorescent particles
probably represented both larger individual and smaller clustered ob-
jects with diameters below the diffraction limit. Thus, it is possible that
the largest PKH26-positive particles imaged in the exosome-containing
samples (Fig. 2b, d) represent aggregated particles. Although there
were ~8-fold more PKH26-positive particles in the exosome-containing
samples compared to the exosome-free controls, the examined char-
acteristics of the PKH26-particles were similar in both samples.
To further characterize the PKH26-positive particles obtained after
staining of the exosome-free controls and exosome-containing samples
using procedure A (Fig. 1), they were analyzed using AF4-MALS. The
AF4 technique has a broad separation range, from a few nanometers up
to a micrometer, and in combination with a MALS detector it is suitable
for separation and characterization of a wide range of biological (e.g.,
virus-like particles, exosomes) and nonbiological (e.g., proteins, plas-
mids, polysaccharides) analytes [53]. Importantly, unlike confocal mi-
croscopy, in addition to the fluorescent particles, AF4-MALS also de-
tects nonfluorescent particles. From the fractograms obtained, the size
distributions, mean sizes, and number densities of the particles were
determined (Fig. 3, Table 1). The sizes were expressed according to two
different radii: root mean square radius (Rrms), and geometric radius
(Rgeom). In addition to the labeled samples, the AF4-MALS analysis was
also performed for the PKH26-staining solution and unstained exo-
somes.
As shown in Fig. 3, the elution profiles of the PKH26-labeled exo-
some-free control (con-procedure A) and PKH26-labeled exosome (exo-
procedure A) samples recorded by MALS showed relatively broad size
distributions (Rrms, 20–100 nm). Interestingly, the elution profile of the
unstained exosomes (exo), which were used as the input sample for the
PKH26-labeling procedures (Fig. 1), mostly coincided with the profiles
of both of the PKH26-labeled samples, so even the exosome-free con-
trol. The presence of similar particles in all three samples was supported
Unstained and unprocessed exosome sample.
b
Number of particles per mL in the final volume of 50 μL DPBS for 20 μg of exosome
input (Fig. 1).
by the mean particle Rrms (Rgeom), of 77 (84) nm and 79 (94) nm for the
PKH26-labeled exosome-free control and PKH26-labeled exosome
samples, respectively, which were slightly higher in the unstained
exosome sample, at 85 (101) nm (Table 1). Importantly, as seen from
Fig. 3a, the PKH26 solution used for the labeling did not give any MALS
signal, which indicates that the concentration of the PKH26-positive
particles before processing (Fig. 1) was below the detection limits.
In addition, the number densities were determined (Fig. 3b,
Table 1). Within the experimental error, the total number density of the
particles in the PKH26-labeled exosome samples
(10.70 × 1010 particles/mL) was 14.3-fold higher than that for the
control samples (0.75 × 1010 particles/mL). This observed higher
number density in the PKH26-labeled exosome samples was not due to
enrichment of any specific particle subpopulation, as a similar increase
in particle numbers was observed over the whole size interval (Fig. 3b).
As also seen by imaging, a small population of larger particles was
detected in the PKH26-labeled exosome samples that was not seen in
the unstained exosomes, which might have represented aggregated
particles (Fig. 3b). To estimate the proportions of exosomes in the
PKH26-labeled exosome samples, we first determined the percent re-
covery of unstained exosomes processed as in procedure A, by mea-
suring the number densities of exosomes before (theoretical yield;
3.85 × 1010 particles/mL, Table 1) and after (experimental yield;
1.19 × 1010 particles/mL, data not shown) the processing, using AF4-
MALS. The recovery of the exosomes exposed to the ultracentrifugation-
based staining of procedure A was approximately 31%. Thus when
staining 20 μg of exosomes (corresponding to 19.25 × 1012 exosomes)
using procedure A (Fig. 1), we expect to obtain approximately 6 × 1012
exosomes in the PKH26-labeled exosome samples. As we detected
53.5 × 1012 particles (10.70 × 1010 particles/mL in 50 μL; Table 1), the
exosomes appear to represent only 11% of all of the particles, with the
rest likely representing PKH26 dye particles (PKH26 nanoparticles;

Fig. 3. AF4-MALS analysis of PKH26-labeled exosome-free controls and exosome samples prepared using procedure A.
Normalized AF4-MALS fractograms of the PKH26-labeling solution (PKH26; grey), PKH26-labeled exosome-free control (con-procedure A; red), unstained exosomes (exo; black) and
PKH26-labeled exosome sample (exo-procedure A; blue) for Rrms (a) and number density (b). Samples were stained following procedure A (Fig. 1). Continuous curves represent
normalized light scattering signal, while empty circles represent Rrms (a) and number density (b).

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P. Pužar Dominkuš et al. BBA - Biomembranes 1860 (2018) 1350–1361

47.5 × 1012).
Similar to the confocal imaging, the AF4-MALS analysis allowed the
detection of ~14-fold more PKH26-positive particles in the exosome-
containing samples compared to the exosome-free controls, with par-
ticles from both samples having similar characteristics. Importantly, in
procedure A, only one out of every 10 particles in the PKH26-labeled
exosome samples appears to represent exosomes. Overall, the com-
monly used ultracentrifugation-based exosome staining of procedure A
generates numerous PKH26 nanoparticles, which are almost indis-
tinguishable from the PKH26-labeled exosomes in terms of their size,
surface area, and fluorescence intensity.

3.2. Low PKH26-positive particle recovery is characteristic of filtration-

based exosome staining

As an alternative, Lässer et al. [21] used a filtration-based exosome

staining procedure, with the concentration and washing of the exo-
somes through 300-kDa filters. To evaluate the efficiency of this
staining procedure and its propensity to promote PKH26 nanoparticles
formation, exosomes and particle-free DPBS (control) were labeled with
PKH26 following procedure B (Fig. 1). The PKH26-positive particles in
the suspension were then evaluated using confocal microscopy, as
above (Fig. 4a–d). Procedure B resulted in the preparation of samples
with similarly low numbers of fluorescent particles per image for both
the PKH26-labeled exosome-free control and PKH26-labeled exosome-
containing samples: 2 ± 2 (n = 10) versus 1 ± 0.1 (n = 20), respec-
tively (Fig. 4e). The particle surface areas and fluorescence intensities
were lower in the PKH26-labeled exosome-free controls than in the
PKH26-labeled exosome-containing samples (Fig. 4f, g). These samples
prepared using procedure B were further analyzed by AF4-MALS, al-
though the sample concentrations were below the detection limit of the
instrument, so no further analyses were performed. Overall, low
PKH26-positive particle recovery was characteristic of the filtration-
based exosome staining of both the exosome-free controls and exosome-
containing samples.

3.3. PKH26 nanoparticles and PKH26-labeled exosomes purified through a

sucrose cushion can be differentiated on the basis of size

Lentiviruses, e.g. human immunodeficiency virus (HIV), and lenti-
viral vectors are generally purified by ultracentrifugation through a
sucrose cushion to remove any impurities of lower buoyant density than
that of virions [54]. Exosomes are similar to HIV in terms of many of
their physical properties and have similar buoyant density in a sucrose
gradient [55,56]. Therefore, we next analyzed whether PKH26 nano-
particles can be removed from PKH26-labeled exosomes using pur-
ification through a sucrose cushion (Fig. 1, procedure C). The PKH26-
labeled exosome-free controls and PKH26-labeled exosome-containing
samples were first analyzed for the numbers of particles per image,
surface areas, and fluorescence intensities using confocal microscopy
(Fig. 5). When compared to the ultracentrifugation-based staining
procedure (procedure A), sucrose cushion ultracentrifugation resulted
in fewer PKH26-positive particles in both the exosome-free controls and
exosome-containing samples, with the large PKH26-labeled fluorescent
aggregates no longer seen in the PKH26-labeled exosome samples
(compare Fig. 5a–d with Fig. 2a–d). The number of PKH26-positive
particles per image for the exosome-free controls was 46 ± 3 (n = 20)
versus 340 ± 6 (n = 20) PKH26-positive particles per image for the
exosome-containing samples (Fig. 5e). Similarly, the PKH26-positive
particle surface area (Fig. 5f) was smaller in the control than in exo-
some-containing samples, whereas the PKH26-positive particle fluor-
escence intensity (Fig. 5g) was similar in both samples prepared using
procedure C. Importantly, there were ~7-fold more PKH26-positive
particles in the exosome-containing samples compared to the exosome-
free controls, and these also differed in their surface areas.
As shown in Fig. 6, sucrose-cushion-purified PKH26-labeled
Fig. 4. PKH26-labeled exosome-free controls and exosome samples prepared using pro-
cedure B.
(a–d) Representative confocal images of PKH26-positive particles in a PKH26-labeled
exosome-free control (i.e., particle-free DPBS; con) (a) and in a PKH26-labeled exosome
(exo) (b) sample prepared following procedure B. Inset: Magnified selected PKH26-po-
sitive particle. Scale bars: 2 μm; inset, 0.4 μm. (c, d) Black and white image masks from
(a), (b) after image segmentation for individual PKH26-positive particles (encircled), as
exosome-free control (c; 1 particle) and exosome (d; 1 particle) samples, respectively.
Scale bars: 2 μm. (e–g) Quantification of the PKH26-positive particles per image field of
2030 μm2, as the numbers (e; N°), surface areas (f; S), and fluorescence intensities (g; Int.).
Data are means ± SEM. Numbers (bottom, e) indicate confocal images analyzed,
whereas numbers (bottom, f) indicate PKH26-positive particles analyzed in f and g. **,
p < 0.01; ***, p < 0.001 versus control (con; Mann-Whitney U tests).
exosome-free control (con-procedure C) and PKH26-labeled exosome-
containing (exo-procedure C) samples were further analyzed by AF4-
MALS for their particle sizes and number densities (Table 1). The
fractograms for the unstained and PKH26-labeled exosome samples
showed relatively broad size distributions (Rrms, 25–100 nm), whereas
the size distribution in the exosome-free control samples was a little
narrower (Rrms, 25–80 nm) (Fig. 6a). The presence of smaller particles
in the PKH26-labeled exosome-free control samples compared to the
PKH26-labeled exosome samples was supported by the mean particle
Rrms (Rgeom), which were 65 (70) nm and 81 (93) nm, respectively
(Table 1). Interestingly, the mean particle size in the PKH26-labeled
exosome samples was similar to the mean size of the unstained exo-
somes, as 85 (101) nm. Although the total number densities of the
particles in the PKH26-labeled exosome-free controls and PKH26-

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P. Pužar Dominkuš et al.
Fig. 5. PKH26-labeled exosome-free controls and exosome samples prepared using pro-
cedure C.
(a–d) Representative confocal images of PKH26-positive particles in a PKH26-labeled
exosome-free control (i.e., particle-free DPBS; con) (a) and in a PKH26-labeled exosome
(exo) (b) sample prepared following procedure C. Inset: Magnified selected PKH26-po-
sitive particle. Scale bars: 2 μm; inset, 0.4 μm. (c, d) Black and white image masks from
(a), (b) after image segmentation for individual PKH26-positive particles, as exosome-free
control (c; 40 particles) and exosome (d; 337 particles) samples, respectively. Scale bars:
2 μm. (e–g) Quantification of the PKH26-positive particles per image field of 2030 μm2, as
the numbers (e; N°), surface areas (f; S), and fluorescence intensities (g; Int.). Data are
means ± SEM. Numbers (bottom, e) indicate confocal images analyzed, whereas num-
bers (top, f) indicate PKH26-positive particles analyzed in f and g. ***, p < 0.001 versus
control (con; Mann-Whitney U tests).
labeled exosome samples were similar (0.79 × 1010 particles/mL vs.
1.13 × 1010 particles/mL, respectively; Table 1), the population of
larger particles was enriched in the PKH26-labeled exosome samples
compared to the PKH26-labeled exosome-free controls (Fig. 6b). Ad-
ditionally, the recovery of unstained exosomes processed using sucrose-
cushion-based staining of procedure C was calculated from the AF4-
MALS measured number densities of exosomes before (theoretical yield;
3.85 × 1010 particles/mL, Table 1) and after (experimental yield;
0.39 × 1010 particles/mL, data not shown) the processing, to be ~10%.
Thus when staining 20 μg of exosomes (corresponding to 19.25 × 1012
exosomes) using procedure C (Fig. 1), we expect to obtain approxi-
mately 1.9 × 1012 exosomes in the PKH26-labeled exosome samples. As
we detected 5.65 × 1012 particles (1.13 × 1010 particles/mL in 50 μL;
Table 1) in those samples, the exosomes appear to represent only 34%
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BBA - Biomembranes 1860 (2018) 1350–1361
of all of the particles, with the rest likely representing PKH26 nano-
particles (3.73 × 1012).
Analysis by AF4-MALS supported the confocal imaging observation
that the PKH26 nanoparticles were smaller in size when compared to
the PKH26-positive particles in the exosome samples. Conversely, only
1.4-fold more PKH26-positive particles were detected in the exosome-
containing samples, compared to the exosome-free control samples.
Three to four out of 10 PKH26-positive particles in the PKH26-labeled
exosome samples appeared to represent exosomes. Importantly, similar
observations were made when using the PKH67 dye instead of the
PKH26 dye for sucrose-cushion-based staining of exosomes (for details
see Appendix A, Table S1, Fig. S1).
In conclusion, although the purification through the sucrose cushion
is not very efficient in removing the PKH26 nanoparticles from the
PKH26-labeled exosome samples, it affects the PKH26 nanoparticle
size, and thereby helps in their differentiation from the PKH26-labeled
exosomes, especially when analyzed using confocal microscopy.
3.4. PKH26-labeled exosomes separate from PKH26 nanoparticles by
passing into denser sucrose gradient fractions
As none of the above procedures provided pure PKH26-labeled
exosomes devoid of PKH26 nanoparticles, the PKH26-labeling protocol
optimized for flow cytometry analysis by Van der Vlist et al. [45] was
then followed. In this procedure, the particles in the PKH26-labeled
exosome samples were separated on a sucrose gradient based on their
buoyant densities, with the PKH26-labeled exosomes generally separ-
ating into fractions with densities between 1.08 g/mL and 1.22 g/mL
[15,57,58]. After following the PKH26-labeling protocol of procedure D
(Fig. 1), fractions 3–5 of the gradient were visibly stained in the PKH26-
labeled exosome-free control (con) and fraction 9 in the PKH26-labeled
exosome-containing sample (exo; data not shown). To isolate the
PKH26-particles from the stained fractions for analysis by confocal
microscopy (Fig. 7), fractions 3–6 (1.08–1.15 g/mL, as measured using
a refractometer) and 7–10 (1.17–1.23 g/mL) were collected for both
samples. These were diluted in a large volume of DPBS and the particles
were pelleted by ultracentrifugation. In the lighter fractions of the su-
crose gradient (i.e., fractions 3–6), the PKH26-labeled exosome-free
controls (Fig. 7a–e, con 1) and PKH26-labeled exosome samples
(Fig. 7a–e, exo 1) contained low and similar numbers of PKH26-positive
particles with similar surface areas and fluorescence intensities (Fig. 7f,
g). However, in the denser fractions of the sucrose gradient (i.e., frac-
tions 7–10), many PKH26-positive particles were observed in the
PKH26-labeled exosome samples (Fig. 7h-l, exo 2), while only a few
PKH26-positive particles were seen in the exosome-free controls
(Fig. 7h-l, con 2). In the PKH26-labeled exosome-free control, the
number of PKH26-positive particles per image was one-tenth than of
the PKH26-labeled exosome sample, as 1 ± 0.3 (n = 19) versus
10 ± 2 (n = 20), respectively (Fig. 7l). Again, the PKH26-positive
particles in both samples had comparable surface areas and fluores-
cence intensities (Fig. 7m, n), which were similar to those reported for
procedure C (Fig. 5f, g). The PKH26-positive particles that were sepa-
rated into the lighter fractions thus appear to represent PKH26 nano-
particles, while those separated into the denser fractions appear to re-
present pure PKH26-labeled exosomes. Samples prepared using
procedure D were further analyzed by AF4-MALS, although as the
concentration of PKH26-positive particles was below the detection limit
of the instrument, no further analyses were performed.
Altogether, these data indicate that procedure D, which includes
separation on a sucrose gradient and pelleting of the PKH26-positive
particles by ultracentrifugation, can be used to efficiently separate
PKH26-labeled exosomes from PKH26 nanoparticles, although this is
achieved at the expense of low PKH26-labeled exosome recovery.
P. Pužar Dominkuš et al.
Fig. 6. AF4-MALS analysis of PKH26-labeled exosome-free and exosome samples prepared using procedure C.
Normalized AF4-MALS fractograms of the PKH26-labeling solution (PKH26), PKH26-labeled exosome-free control (con-procedure C; red), unstained exosomes (exo; black) and PKH26-
labeled exosome sample (exo-procedure C; blue) for Rrms (a) and number density (b). Samples were stained following procedure C (Fig. 1). Continuous curves represent normalized light
scattering signal, while empty circles represent Rrms (a) and number density (b).
3.5. PHK26 nanoparticles are internalized into cultured astrocytes
Characterization of PKH26-positive particles in PKH26-labeled
samples revealed that PKH26 nanoparticles are present in the exosome-
free controls, whereas a mix of PKH26 nanoparticles and PKH26-la-
beled exosomes are present in the PKH26-labeled exosome-containing
samples. To investigate the physiological relevance of contaminating
PKH26 nanoparticles in exosome cell targeting, we tested whether
cultured astrocytes can pick-up and internalize PKH26 nanoparticles
obtained using procedure C (con) in a way similar to that seen for
PKH26-positive particles present in the PKH26-labeled exosome-con-
taining samples (exo). Confocal microscopy of live cells (z-stacked)
revealed that both PKH26 nanoparticles (Fig. 8a) and PKH26-positive
particles of the exosome-containing samples (Fig. 8b) localized to si-
milar subcellular compartments, and were visible as numerous
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BBA - Biomembranes 1860 (2018) 1350–1361
fluorescent puncta in the cell cytoplasm. After 3 h co-incubation of the
cells with PKH26 nanoparticles (con) and PKH26-positive particles of
the exosome-containing samples (exo) at 37 °C, the fluorescent com-
partments per cell were 127 ± 24 (n = 12) and 662 ± 78 (n = 12),
respectively (Fig. 8c). This 5.2-fold difference in the number of in-
tracellular fluorescent compartments across these two sets of labeled
cells closely mirrors the ~7-fold difference in the number of PKH26-
positive particles in the PKH26-labeled exosome-free controls and the
PKH26-labeled exosome samples loaded on the astrocytes. Interest-
ingly, the mean volume of these fluorescent compartments that picked-
up the PKH26 nanoparticles (con) was ~76% that of the compartments
that picked-up the PKH26-positive particles of the exosome-containing
sample (exo; Fig. 8d), whereas the mean compartment fluorescence
intensity was similar in these cells (Fig. 8e).
These intracellular fluorescent compartments are likely endocytotic
Fig. 7. Purified PKH26-labeled exosome-free
controls and exosome samples prepared using
procedure D.
(a–d, h–k) Representative confocal images of
PKH26-positive particles isolated from the lighter
(fractions 3–6) (a, b) and denser (fractions 7–10)
(h, i) sucrose gradient fractions after separation of
PKH26-labeled exosome-free control (a, con 1; h,
con 2) and PKH26-labeled exosome (b, exo 1; i,
exo 2) samples. Inset: Magnified selected PKH26-
positive particle. Scale bars: 2 μm; inset, 0.4 μm.
(c, d, j, k) Black and white image masks from (a),
(b), (h), (i) after image segmentation for in-
dividual PKH26-positive particles (encircled) as
exosome-free control (c, j) and exosome (d, k)
samples. Scale bars: 2 μm. (e–g, l–n)
Quantification of the PKH26-positive particles per
image field of 2030 μm2, as the numbers (e, l; N°),
surface areas (f, m; S), and fluorescence in-
tensities (g, n; Int.). Data are means ± SEM.
Numbers (bottom, e and l) indicate confocal
images analyzed, whereas numbers (bottom, f
and m) indicate PKH26-positive particles ana-
lyzed in f and g or m and n, respectively. ***,
p < 0.001 versus control (con; Mann-Whitney U
tests).
P. Pužar Dominkuš et al.
Fig. 8. Internalization of PKH26 nanoparticles and PKH26-positive particles of the exo-
some-containing samples into subcellular compartments of cultured astrocytes.
(a, b) Representative three-dimensional shaded display of individual live cultured as-
trocytes that internalized PKH26 nanoparticles (a, con) and PKH26-positive particles
present in the PKH26-labeled exosome-containing samples (b, exo) into intracellular
compartments, observed as numerous bright fluorescent puncta. Scale bars, 10 μm. (c–e)
Quantification of the number (c; N°), volume (d; V), and fluorescence intensities (e; Int.)
of compartments (Comp.) that internalized PKH26 nanoparticles (con) and PKH26-posi-
tive particles of the exosome-containing samples (exo) per cell. Data are means ± SEM.
Numbers (bottom, c) indicate cells analyzed, whereas numbers (top, d) indicate PKH26-
positive compartments analyzed in d and e. ***, p < 0.001 versus control (con; Mann-
Whitney U tests). (f-g) Confocal micrographs of double-labeled live astrocytes that in-
ternalized PKH26 nanoparticles (f, con, left) or PKH26-positive particles present in the
PKH26-labeled exosome-containing samples (g, exo, left) and fluorescent dextran (Dex,
middle) into the common subcellular compartments as revealed by white mask images
displaying co-localized pixels (Mask, right). Scale bars: 10 μm.
vesicles, as we observed a pattern of internalization that is typical for
normal membrane recycling. To test this, we microscopically examined
cells that were simultaneously exposed either to PKH26 nanoparticles
or to PKH26-positive particles present in the PKH26-labeled exosome-
containing sample, and to 20 μM fluorescent dextran. After 3 h co-in-
cubation of cells with aforementioned probes, ample fluorescence co-
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BBA - Biomembranes 1860 (2018) 1350–1361
localization was observed between the PKH26-labeled fluorescent
compartments and the dextran-laden vesicles (Fig. 8f, g). These data
indicate that the PKH26 nanoparticles present in the PKH26-labeled
exosome-free controls internalized into similar astrocytic compartments
as the mix of PKH26 nanoparticles and PKH26-labeled exosomes pre-
sent in PKH26-labeled exosome-containing samples.
4. Discussion
Despite the wide use of PKH-labeled EVs in cell targeting and
functional studies [13,14], the nonEV-associated fluorescent structures
detected by Lai et al. [46] have never been systematically character-
ized, and their internalization by cells has never been evaluated. Here,
we have shown for the first time that numerous PKH26 nanoparticles
are formed during ultracentrifugation-based exosome staining, and that
these are almost indistinguishable from PKH26-labeled exosomes in
terms of their size, surface area, and fluorescence intensity, as shown by
confocal imaging and AF4-MALS analysis. When PKH26-labeled exo-
some samples were purified through sucrose, the PKH26 nanoparticles
were differentiated from the PKH26-labeled exosomes based on their
reduced size. However, these PKH26 nanoparticles can only be physi-
cally removed from the PKH26-labeled exosome samples when they are
separated on a sucrose gradient. Of note, this is achieved at the expense
of low PKH26-labeled exosome recovery. Importantly, PKH26 nano-
particles can also be internalized into primary astrocytes into similar
subcellular compartments as seen for PKH26-positive particles of the
PKH26-labeled exosome-containing samples.
Regardless of the staining procedure used, PKH26-positive particles
were detected in the PKH26-labeled exosome-free controls, although to
different extents. Around 0.75 × 1010 PKH26 particles/mL were de-
tected in the PKH26-labeled exosome-free controls after ultra-
centrifugation-based staining of procedure A, as determined using AF4-
MALS. Similar levels were detected even after purification through a
sucrose cushion (0.79 × 1010 PKH26 particles/mL) when following
procedure C. In comparison, the particle numbers were below the de-
tection limit of the AF4-MALS analysis following purification of the
PKH26-positive particles of the exosome-free control on a sucrose
gradient according to procedure D, as they were greatly decreased (by
~97% as assessed by confocal imaging). Importantly, the sizes (i.e.,
Rrms, Rgeom), surface areas, and fluorescence intensities of the PKH26-
positive particles in the exosome-free control samples were mostly si-
milar to the characteristics of the PKH26-positive particles in the exo-
some-containing samples. Of interest, the PKH26-positive particles in
the ultracentrifugation-based PKH26-labeled exosome-free controls of
procedure A showed a broad size distribution (Rrms, 20–100 nm; mean
Rrms [Rgeom], 77 [84] nm), which mirrored the particle sizes in the
PKH26-labeled exosome samples. Only when the PKH26-positive par-
ticles were purified through the hyperosmotic sucrose cushion of pro-
cedure C did those in the exosome-free control samples become more
homogenous in size, with a smaller mean Rrms (Rgeom) of 65 (70) nm.
This will probably be due to their loss of water [59]. All of these details
indicate that the PKH26 nanoparticles in the exosome-free control
samples could represent PKH26 micelles, which in general cannot be
differentiated from the PKH26-labeled exosomes in terms of the char-
acteristics that were measured here.
PKH26 nanoparticles were also detected in the PKH26-labeled
exosome samples regardless of the staining procedure used. For the
ultracentrifugation-based staining of procedure A, the PKH26-labeled
exosomes represented only 11% of all of the PKH26-positive particles
detected in the PKH26-labeled exosome samples, with the rest re-
presenting PKH26 nanoparticles. Thus, there were 13-fold more PKH26
nanoparticles in the PKH26-labeled exosome samples compared to the
corresponding exosome-free control for the ultracentrifugation based
procedure A. This implies that the PKH26 nanoparticle numbers in the
PKH26-labeled exosome samples cannot simply be inferred from their
numbers in the corresponding PKH26-labeled exosome-free control
P. Pužar Dominkuš et al.
samples. Similarly we showed that for the sucrose-cushion-based
staining of procedure C, exosomes appear to represent 34% of all of the
PKH26-positive particles in the PKH26-labeled exosome samples, with
the rest of these representing PKH26 nanoparticles. Thus comparable
numbers of PKH26 nanoparticles are present in the PKH26-labeled
exosome samples and exosome-free controls for the sucrose-cushion-
based procedure C. Contrary to previously published data [60,61], low
PKH26-positive particle recovery was characteristic of the filtration-
based exosome staining of procedure B for both the exosome-free
controls and exosome-containing samples. This discrepancy might be
due to the nature of the filtration membranes, as a concentrating device
that contains a polyethersulfone membrane can reduce EV yield by at
least two-thirds compared to one that contains a cellulose membrane
[21,61]. The lower propensity of the PKH26 dye to form particles when
using non-sedimentation processes for staining might also contribute
here. Importantly, by using the sucrose gradient of procedure D it was
possible to separate PKH26 nanoparticles from PKH26-labeled exo-
somes in the PKH26-labeled exosome samples. However, the separation
is achieved at the expense of < 2% recovery of the PKH26-labeled
exosomes, as determined here by confocal imaging. Similarly, a study
by Van Deun et al. [62] showed that about two-thirds fewer exosomes
were recovered by an iodixanol-gradient-based isolation procedure
compared to ultracentrifugation.
The present data support the sucrose-gradient-based staining of
procedure D as the method of choice to obtain PKH26-labeled exosomes
that are devoid of PKH26 nanoparticles for use in EV uptake and
functional studies. As PKH26 nanoparticles only contain buffer, they
have lower buoyance density in comparison to PKH26-labeled exo-
somes, and therefore they can be separated into lighter fractions of a
sucrose gradient. The PKH26-labeled exosomes separated most promi-
nently into fraction 9, with a density of 1.21 g/mL, which is at the edge
of the density interval given previously as 1.08 g/mL to 1.22 g/mL
[15,57,58], which is typical for unstained exosomes. In other studies,
stained EVs floated at sucrose densities of 1.11 g/mL to 1.19 g/mL
[15,63]; however, smaller EV populations might have been missed
here, as the detection limit of flow cytometry is 100 nm in diameter.
Interestingly, there was no severe effect of sucrose on exosome size,
with the mean Rrms (Rgeom) and surface areas of the PKH26-positive
particles in the PKH26-labeled exosome samples remaining at 81 (93)
nm and 0.14 μm2, respectively. Although some concerns have been
expressed about the effects of sucrose on the biological functions of EVs,
Zech et al. [64] did not observe impairment of leukocyte effector
functions for sucrose-gradient-enriched compared to 0.2-μm-filtered
exosomes from a pancreatic adenocarcinoma. As determined by several
biochemical and biophysical techniques, use of sucrose and iodixanol
gradients also represents an efficient way to obtain highly pure exo-
somes from serum, and these showed higher biological activity in terms
of NFκB nuclear translocation compared to ultracentrifugation-based
and PEG-based exosome preparations [44]. Importantly, lentiviruses,
like HIV, and lentiviral vectors, can also be purified by ultra-
centrifugation through a sucrose cushion for further use in in vitro and
in vivo infection studies [54].
The presence of PKH26 nanoparticles in PKH26-labeled EV samples
has never been accounted for in previous EV functional studies.
Although they do not carry any functional molecules, PKH26 nano-
particles might contribute to the labeling of target cells and affect es-
timation of the cellular uptake of PKH26-labeled exosomes. Here, we
have shown that PKH26 nanoparticles are indeed internalized into
primary astrocytes in similar compartments as seen for PKH26-positive
particles of the PKH26-labeled exosome-containing samples. Ample
fluorescence co-localization between the PKH26-laden fluorescent
compartments and the dextran-laden vesicles revealed that PKH26-
positive particles likely internalized into astrocytic endo-/lysosomes
[65]. This observation is of great importance, as only one or three out of
10 PKH26-positive particles actually represent PKH26-labeled exo-
somes in the PKH26-labeled exosome samples stained following
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BBA - Biomembranes 1860 (2018) 1350–1361
procedure A and C, respectively. Thus PKH26-labeled exosome samples
are mostly composed of PKH26 nanoparticles. Consequently, it is cru-
cial to remove PKH26 nanoparticles from PKH26-labeled exosomes
before performing cell targeting and internalization studies.
At present, we can only speculate on the mechanisms of the cellular
uptake of PKH26 nanoparticles. EVs can be taken up by cells through a
variety of receptor-dependent pathways, or they can fuse with or just
bind to the plasma membrane [40–42]. Importantly, EVs can also be
internalized through receptor-independent mechanisms, like macro-
pinocytosis, where extracellular fluid and its components are enveloped
by an invagination and there is fusion of the membrane ruffles, to form
a vesicular bud that pinches off into the cytosol [66]. This was sup-
ported by Fitzner et al. [67], who showed that sucrose-gradient-purified
PKH67-labeled exosomes from oligodendrocytes are selectively trans-
ferred to microglia by macropinocytosis. Macropinocytosis can be
constitutive, as is found in microglia [67,68], macrophages [69], and
dendritic [70] and cancer cells [68,71], or it can be growth-factor-ac-
tivated. Another study postulated that this process is present in most
cells, with the exception of the brain microvessel endothelial cells, and
potentially some other [72]. Importantly, macropinocytosis is also one
of the pathways that can be exploited for cellular uptake of nanome-
dicines, including polymeric micelles [72]. Thus we can assume that
PKH26 nanoparticles can be taken up by cells through macro-
pinocytosis, a process that can be promoted further by the strong
electrostatic interaction between the positively charged carbocyanine
(PKH) dye and the negatively charged plasma membrane, as was shown
to be the case for cationic nanoparticles [72,73].
Interestingly, a recent study showed that Lipofectamine, which is a
reagent composed of lipid subunits that can form cationic liposomes,
can increase internalization of exosomes containing tau seeds through
induction of the binding of EVs and formation of EV clusters [74]. In
another study, exosomes released from HIV-infected cells were shown
to render resting CD4+ T lymphocytes permissive to virus internaliza-
tion and infection [36,75]. Similarly, the PKH26 nanoparticles in
PKH26-labeled exosome samples might promote cellular uptake of
PKH26-labeled exosomes, through changing the exosome internaliza-
tion characteristics. In addition to internalization of PKH26-positive
particles into astrocytic subcellular compartments, the cellular fluor-
escent patterns observed in the present study might also be the result of
nonspecific leakage of the dye molecules from PKH26-positive particles
to the plasma membrane, and subsequently to cellular membranes, as
this would lead to a pattern of internalization similar to that of normal
membrane recycling [42]. However, several studies have shown that
passive dye transfer is probably a minor process, as fixed microglia did
not stain with PKH67 [67] and low temperature and/or excess un-
labeled exosomes reduced the uptake of glioblastoma cells [76], en-
dothelial cells [44], and bladder cancer cells [14].
Altogether, PKH26 staining of EVs can be an informative tool, but
only when an appropriate isolation method is used to remove PKH26
nanoparticles, and rigorous controls are used to evaluate EV uptake by
target cells. Our observations about the characteristics of the PKH26
dye can be extended to the PKH67 dye, as shown here by us (Appendix
A), and likely to the other lipophilic long-chain carbocyanines like DiO
and Dil, as PKH26 is structurally identical to DilC18 [77]. The char-
acteristics of alternative membrane dyes used for staining of EVs, like
phospholipid dye rhodamine-DHPE [78] and styryl dye FM 1–43 [79],
would need to be further studied.
Competing interests
The authors declare no conflict of interest and funding.
Author contributions
Conceived and designed the experiments: PPD, MS, RZ, AP, EŽ, MK,
ML.
Performed the experiments: PPD, MS, SS, EL.
Analyzed the data: MS, SS, EL, ML.
P. Pužar Dominkuš et al.
Wrote the paper: PPD, MS, SS, EL, RZ, AP, EŽ, MK, ML.
Transparency document
The http://dx.doi.org/10.1016/j.bbamem.2018.03.013 associated
this article can be found, in online version.
Acknowledgments
This work was supported by the Slovenian Research Agency (ARRS)
[research grants J3-5499, P1-170, P3-310, and P2-0145].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bbamem.2018.03.013.
References
[1] P.K. Wallace, J.D. Tario Jr., J.L. Fisher, S.S. Wallace, M.S. Ernstoff, K.A. Muirhead,
Tracking antigen-driven responses by flow cytometry: monitoring proliferation by
dye dilution, Cytometry A 73 (2008) 1019–1034.
[2] F. Progatzky, M.J. Dallman, C. Lo Celso, From seeing to believing: labelling stra-
tegies for in vivo cell-tracking experiments, Interface Focus 3 (2013) 20130001.
[3] H.E. Ghoneim, P.G. Thomas, J.A. McCullers, Depletion of alveolar macrophages
during influenza infection facilitates bacterial superinfections, J. Immunol. 191
(2013) 1250–1259.
[4] M.E. Sheehy, A.B. McDermott, S.N. Furlan, P. Klenerman, D.F. Nixon, A novel
technique for the fluorometric assessment of T lymphocyte antigen specific lysis, J.
Immunol. Methods 249 (2001) 99–110.
[5] C.R. Parish, Fluorescent dyes for lymphocyte migration and proliferation studies,
Immunol. Cell Biol. 77 (1999) 499–508.
[6] S.A. Bapat, A. Krishnan, A.D. Ghanate, A.P. Kusumbe, R.S. Kalra, Gene expression:
protein interaction systems network modeling identifies transformation-associated
molecules and pathways in ovarian cancer, Cancer Res. 70 (2010) 4809–4819.
[7] M. Schubert, N. Herbert, I. Taubert, D. Ran, R. Singh, V. Eckstein, M. Vitacolonna,
A.D. Ho, M. Zoller, Differential survival of AML subpopulations in NOD/SCID mice,
Exp. Hematol. 39 (2011) 250–263 (e254).
[8] P.J. Hendrikx, C.M. Martens, A. Hagenbeek, J.F. Keij, J.W. Visser, Homing of
fluorescently labeled murine hematopoietic stem cells, Exp. Hematol. 24 (1996)
129–140.
[9] F. Lassailly, E. Griessinger, D. Bonnet, “Microenvironmental contaminations”s in-
duced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell
tracking, Blood 115 (2010) 5347–5354.
[10] P. Li, R. Zhang, H. Sun, L. Chen, F. Liu, C. Yao, M. Du, X. Jiang, PKH26 can transfer
to host cells in vitro and vivo, Stem Cells Dev. 22 (2013) 340–344.
[11] W.E. Samlowski, B.A. Robertson, B.K. Draper, E. Prystas, J.R. McGregor, Effects of
supravital fluorochromes used to analyze the in vivo homing of murine lympho-
cytes on cellular function, J. Immunol. Methods 144 (1991) 101–115.
[12] D.J. Oh, G.M. Lee, K. Francis, B.O. Palsson, Phototoxicity of the fluorescent mem-
brane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell
line, Cytometry 36 (1999) 312–318.
[13] A. Riches, E. Campbell, E. Borger, S. Powis, Regulation of exosome release from
mammary epithelial and breast cancer cells - a new regulatory pathway, Eur. J.
Cancer 50 (2014) 1025–1034.
[14] C.A. Franzen, P.E. Simms, A.F. Van Huis, K.E. Foreman, P.C. Kuo, G.N. Gupta,
Characterization of uptake and internalization of exosomes by bladder cancer cells,
Biomed. Res. Int. 2014 (2014) 619829.
[15] C. Thery, S. Amigorena, G. Raposo, A. Clayton, Isolation and characterization of
exosomes from cell culture supernatants and biological fluids, Current Protocols in
Cell Biology/Editorial Board (2006) Juan S. Bonifacino ..., 3 22 (Chapter 3 (2006)
Unit).
[16] N. Arraud, R. Linares, S. Tan, C. Gounou, J.M. Pasquet, S. Mornet, A.R. Brisson,
Extracellular vesicles from blood plasma: determination of their morphology, size,
phenotype and concentration, J. Thromb. Haemost. 12 (2014) 614–627.
[17] M.G. Harrington, A.N. Fonteh, E. Oborina, P. Liao, R.P. Cowan, G. McComb,
J.N. Chavez, J. Rush, R.G. Biringer, A.F. Huhmer, The morphology and biochem-
istry of nanostructures provide evidence for synthesis and signaling functions in
human cerebrospinal fluid, Cerebrospinal Fluid Res. 6 (2009) 10.
[18] T. Pisitkun, R.F. Shen, M.A. Knepper, Identification and proteomic profiling of
exosomes in human urine, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 13368–13373.
[19] I. Lozano-Ramos, I. Bancu, A. Oliveira-Tercero, M.P. Armengol, A. Menezes-Neto,
H.A. Del Portillo, R. Lauzurica-Valdemoros, F.E. Borras, Size-exclusion chromato-
graphy-based enrichment of extracellular vesicles from urine samples, J Extracell
Vesicles 4 (2015) 27369.
[20] A. Poliakov, M. Spilman, T. Dokland, C.L. Amling, J.A. Mobley, Structural hetero-
geneity and protein composition of exosome-like vesicles (prostasomes) in human
semen, Prostate 69 (2009) 159–167.
[21] C. Lasser, V.S. Alikhani, K. Ekstrom, M. Eldh, P.T. Paredes, A. Bossios, M. Sjostrand,
S. Gabrielsson, J. Lotvall, H. Valadi, Human saliva, plasma and breast milk
1360
BBA - Biomembranes 1860 (2018) 1350–1361
exosomes contain RNA: uptake by macrophages, J. Transl. Med. 9 (2011) 9.
[22] A. Asea, C. Jean-Pierre, P. Kaur, P. Rao, I.M. Linhares, D. Skupski, S.S. Witkin, Heat
shock protein-containing exosomes in mid-trimester amniotic fluids, J. Reprod.
Immunol. 79 (2008) 12–17.
[23] C. Admyre, J. Grunewald, J. Thyberg, S. Gripenback, G. Tornling, A. Eklund,
A. Scheynius, S. Gabrielsson, Exosomes with major histocompatibility complex class
II and co-stimulatory molecules are present in human BAL fluid, Eur. Respir. J. 22
(2003) 578–583.
[24] K. Skriner, K. Adolph, P.R. Jungblut, G.R. Burmester, Association of citrullinated
proteins with synovial exosomes, Arthritis Rheum. 54 (2006) 3809–3814.
[25] J.C. Gross, V. Chaudhary, K. Bartscherer, M. Boutros, Active Wnt proteins are se-
creted on exosomes, Nat. Cell Biol. 14 (2012) 1036–1045.
[26] G. Lachenal, K. Pernet-Gallay, M. Chivet, F.J. Hemming, A. Belly, G. Bodon, B. Blot,
G. Haase, Y. Goldberg, R. Sadoul, Release of exosomes from differentiated neurons
and its regulation by synaptic glutamatergic activity, Mol. Cell. Neurosci. 46 (2011)
409–418.
[27] J. Conde-Vancells, E. Rodriguez-Suarez, N. Embade, D. Gil, R. Matthiesen, M. Valle,
F. Elortza, S.C. Lu, J.M. Mato, J.M. Falcon-Perez, Characterization and compre-
hensive proteome profiling of exosomes secreted by hepatocytes, J. Proteome Res. 7
(2008) 5157–5166.
[28] J. Conde-Vancells, E. Gonzalez, S.C. Lu, J.M. Mato, J.M. Falcon-Perez, Overview of
extracellular microvesicles in drug metabolism, Expert Opin. Drug Metab. Toxicol.
6 (2010) 543–554.
[29] A.E. Morelli, A.T. Larregina, W.J. Shufesky, M.L. Sullivan, D.B. Stolz,
G.D. Papworth, A.F. Zahorchak, A.J. Logar, Z. Wang, S.C. Watkins, L.D. Falo Jr.,
A.W. Thomson, Endocytosis, intracellular sorting, and processing of exosomes by
dendritic cells, Blood 104 (2004) 3257–3266.
[30] P.D. Robbins, A.E. Morelli, Regulation of immune responses by extracellular ve-
sicles, Nat. Rev. Immunol. 14 (2014) 195–208.
[31] A. Janowska-Wieczorek, M. Wysoczynski, J. Kijowski, L. Marquez-Curtis,
B. Machalinski, J. Ratajczak, M.Z. Ratajczak, Microvesicles derived from activated
platelets induce metastasis and angiogenesis in lung cancer, Int. J. Cancer 113
(2005) 752–760.
[32] J. Skog, T. Wurdinger, S. van Rijn, D.H. Meijer, L. Gainche, M. Sena-Esteves,
W.T. Curry Jr., B.S. Carter, A.M. Krichevsky, X.O. Breakefield, Glioblastoma mi-
crovesicles transport RNA and proteins that promote tumour growth and provide
diagnostic biomarkers, Nat. Cell Biol. 10 (2008) 1470–1476.
[33] R. Ji, B. Zhang, X. Zhang, J. Xue, X. Yuan, Y. Yan, M. Wang, W. Zhu, H. Qian, W. Xu,
Exosomes derived from human mesenchymal stem cells confer drug resistance in
gastric cancer, Cell Cycle 14 (2015) 2473–2483.
[34] E. Emmanouilidou, K. Melachroinou, T. Roumeliotis, S.D. Garbis, M. Ntzouni,
L.H. Margaritis, L. Stefanis, K. Vekrellis, Cell-produced alpha-synuclein is secreted
in a calcium-dependent manner by exosomes and impacts neuronal survival, J.
Neurosci. 30 (2010) 6838–6851.
[35] B. Fevrier, D. Vilette, F. Archer, D. Loew, W. Faigle, M. Vidal, H. Laude, G. Raposo,
Cells release prions in association with exosomes, Proc. Natl. Acad. Sci. U. S. A. 101
(2004) 9683–9688.
[36] M. Lenassi, G. Cagney, M. Liao, T. Vaupotic, K. Bartholomeeusen, Y. Cheng,
N.J. Krogan, A. Plemenitas, B.M. Peterlin, HIV Nef is secreted in exosomes and
triggers apoptosis in bystander CD4+ T cells, Traffic 11 (2010) 110–122.
[37] L. Rajendran, M. Honsho, T.R. Zahn, P. Keller, K.D. Geiger, P. Verkade, K. Simons,
Alzheimer's disease beta-amyloid peptides are released in association with exo-
somes, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 11172–11177.
[38] M.P. Zaborowski, L. Balaj, X.O. Breakefield, C.P. Lai, Extracellular vesicles: com-
position, biological relevance, and methods of study, Bioscience 65 (2015)
783–797.
[39] E. van der Pol, F.A. Coumans, A.E. Grootemaat, C. Gardiner, I.L. Sargent,
P. Harrison, A. Sturk, T.G. van Leeuwen, R. Nieuwland, Particle size distribution of
exosomes and microvesicles determined by transmission electron microscopy, flow
cytometry, nanoparticle tracking analysis, and resistive pulse sensing, J. Thromb.
Haemost. 12 (2014) 1182–1192.
[40] D.D. Taylor, C. Gercel-Taylor, The origin, function, and diagnostic potential of RNA
within extracellular vesicles present in human biological fluids, Front. Genet. 4
(2013) 142.
[41] A. Montecalvo, A.T. Larregina, W.J. Shufesky, D.B. Stolz, M.L. Sullivan,
J.M. Karlsson, C.J. Baty, G.A. Gibson, G. Erdos, Z. Wang, J. Milosevic,
O.A. Tkacheva, S.J. Divito, R. Jordan, J. Lyons-Weiler, S.C. Watkins, A.E. Morelli,
Mechanism of transfer of functional microRNAs between mouse dendritic cells via
exosomes, Blood 119 (2012) 756–766.
[42] L.A. Mulcahy, R.C. Pink, D.R. Carter, Routes and mechanisms of extracellular ve-
sicle uptake, J Extracell Vesicles 3 (2014).
[43] C. Barres, L. Blanc, P. Bette-Bobillo, S. Andre, R. Mamoun, H.J. Gabius, M. Vidal,
Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates
vesicle uptake by macrophages, Blood 115 (2010) 696–705.
[44] G. Di Noto, M. Chiarini, L. Paolini, E.L. Mazzoldi, V. Giustini, A. Radeghieri,
L. Caimi, D. Ricotta, Immunoglobulin free light chains and GAGs mediate multiple
myeloma extracellular vesicles uptake and secondary NfkappaB nuclear transloca-
tion, Front. Immunol. 5 (2014) 517.
[45] E.J. van der Vlist, E.N. Nolte-'t Hoen, W. Stoorvogel, G.J. Arkesteijn, M.H. Wauben,
Fluorescent labeling of nano-sized vesicles released by cells and subsequent quan-
titative and qualitative analysis by high-resolution flow cytometry, Nat. Protoc. 7
(2012) 1311–1326.
[46] C.P. Lai, E.Y. Kim, C.E. Badr, R. Weissleder, T.R. Mempel, B.A. Tannous,
X.O. Breakefield, Visualization and tracking of tumour extracellular vesicle delivery
and RNA translation using multiplexed reporters, Nat. Commun. 6 (2015) 7029.
[47] E. Pawelczyk, E.K. Jordan, A. Balakumaran, A. Chaudhry, N. Gormley, M. Smith,
P. Pužar Dominkuš et al.
B.K. Lewis, R. Childs, P.G. Robey, J.A. Frank, In vivo transfer of intracellular labels
from locally implanted bone marrow stromal cells to resident tissue macrophages,
PLoS One 4 (2009) e6712.
[48] B. Sumer, J. Gao, Theranostic nanomedicine for cancer, Nanomedicine (London) 3
(2008) 137–140.
[49] T. Katsuda, R. Tsuchiya, N. Kosaka, Y. Yoshioka, K. Takagaki, K. Oki, F. Takeshita,
Y. Sakai, M. Kuroda, T. Ochiya, Human adipose tissue-derived mesenchymal stem
cells secrete functional neprilysin-bound exosomes, Sci. Rep. 3 (2013) 1197.
[50] N. Kol, M. Tsvitov, L. Hevroni, S.G. Wolf, H.B. Pang, M.S. Kay, I. Rousso, The effect
of purification method on the completeness of the immature HIV-1 gag shell, J.
Virol. Methods 169 (2010) 244–247.
[51] C. Gardiner, Y.J. Ferreira, R.A. Dragovic, C.W. Redman, I.L. Sargent, Extracellular
vesicle sizing and enumeration by nanoparticle tracking analysis, J. Extracellular
Vesicles 2 (2013).
[52] J.P. Schwartz, D.J. Wilson, Preparation and characterization of type 1 astrocytes
cultured from adult rat cortex, cerebellum, and striatum, Glia 5 (1992) 75–80.
[53] S. Sitar, A. Kejzar, D. Pahovnik, K. Kogej, M. Tusek-Znidaric, M. Lenassi, E. Zagar,
Size characterization and quantification of exosomes by asymmetrical-flow field-
flow fractionation, Anal. Chem. 87 (2015) 9225–9233.
[54] G. Tiscornia, O. Singer, I.M. Verma, Production and purification of lentiviral vec-
tors, Nat. Protoc. 1 (2006) 241–245.
[55] R. Cantin, J. Diou, D. Belanger, A.M. Tremblay, C. Gilbert, Discrimination between
exosomes and HIV-1: purification of both vesicles from cell-free supernatants, J.
Immunol. Methods 338 (2008) 21–30.
[56] M. Dettenhofer, X.F. Yu, Highly purified human immunodeficiency virus type 1
reveals a virtual absence of vif in virions, J. Virol. 73 (1999) 1460–1467.
[57] G. Raposo, H.W. Nijman, W. Stoorvogel, R. Liejendekker, C.V. Harding, C.J. Melief,
H.J. Geuze, B lymphocytes secrete antigen-presenting vesicles, J. Exp. Med. 183
(1996) 1161–1172.
[58] C. Thery, L. Zitvogel, S. Amigorena, Exosomes: composition, biogenesis and func-
tion, Nat. Rev. Immunol. 2 (2002) 569–579.
[59] S. Hupfeld, H.H. Moen, D. Ausbacher, H. Haas, M. Brandl, Liposome fractionation
and size analysis by asymmetrical flow field-flow fractionation/multi-angle light
scattering: influence of ionic strength and osmotic pressure of the carrier liquid,
Chem. Phys. Lipids 163 (2010) 141–147.
[60] H.G. Lamparski, A. Metha-Damani, J.Y. Yao, S. Patel, D.H. Hsu, C. Ruegg, J.B. Le
Pecq, Production and characterization of clinical grade exosomes derived from
dendritic cells, J. Immunol. Methods 270 (2002) 211–226.
[61] R.J. Lobb, M. Becker, S.W. Wen, C.S.F. Wong, A.P. Wiegmans, A. Leimgruber,
A. Moller, Optimized exosome isolation protocol for cell culture supernatant and
human plasma, J Extracell Vesicles 4 (2015).
[62] J. Van Deun, P. Mestdagh, R. Sormunen, V. Cocquyt, K. Vermaelen,
J. Vandesompele, M. Bracke, O. De Wever, A. Hendrix, The impact of disparate
isolation methods for extracellular vesicles on downstream RNA profiling, J
Extracell Vesicles 3 (2014).
[63] I. Nazarenko, A.K. Rupp, P. Altevogt, Exosomes as a potential tool for a specific
delivery of functional molecules, Methods Mol. Biol. 1049 (2013) 495–511.
[64] D. Zech, S. Rana, M.W. Buchler, M. Zoller, Tumor-exosomes and leukocyte acti-
vation: an ambivalent crosstalk, Cell Commun. Signal. 10 (2012) 37.
[65] N. Vardjan, M. Gabrijel, M. Potokar, U. Svajger, M. Kreft, M. Jeras, Y. de Pablo,
1361
BBA - Biomembranes 1860 (2018) 1350–1361
M. Faiz, M. Pekny, R. Zorec, IFN-gamma-induced increase in the mobility of MHC
class II compartments in astrocytes depends on intermediate filaments, J.
Neuroinflammation 9 (2012) 144.
[66] G.J. Doherty, H.T. McMahon, Mechanisms of endocytosis, Annu. Rev. Biochem. 78
(2009) 857–902.
[67] D. Fitzner, M. Schnaars, D. van Rossum, G. Krishnamoorthy, P. Dibaj, M. Bakhti,
T. Regen, U.K. Hanisch, M. Simons, Selective transfer of exosomes from oligoden-
drocytes to microglia by macropinocytosis, J. Cell Sci. 124 (2011) 447–458.
[68] K. Yuyama, H. Sun, S. Mitsutake, Y. Igarashi, Sphingolipid-modulated exosome
secretion promotes clearance of amyloid-beta by microglia, J. Biol. Chem. 287
(2012) 10977–10989.
[69] D. Feng, W.L. Zhao, Y.Y. Ye, X.C. Bai, R.Q. Liu, L.F. Chang, Q. Zhou, S.F. Sui,
Cellular internalization of exosomes occurs through phagocytosis, Traffic 11 (2010)
675–687.
[70] Z. Liu, P.A. Roche, Macropinocytosis in phagocytes: regulation of MHC class-II-re-
stricted antigen presentation in dendritic cells, Front. Physiol. 6 (2015) 1.
[71] C. Escrevente, S. Keller, P. Altevogt, J. Costa, Interaction and uptake of exosomes by
ovarian cancer cells, BMC Cancer 11 (2011) 108.
[72] L. Kou, J. Sun, Y. Zhai, Z. He, The endocytosis and intracellular fate of nanome-
dicines: implication for rational design, Asian J. Pharm. Sci. 8 (2013) 1–10.
[73] O. Harush-Frenkel, E. Rozentur, S. Benita, Y. Altschuler, Surface charge of nano-
particles determines their endocytic and transcytotic pathway in polarized MDCK
cells, Biomacromolecules 9 (2008) 435–443.
[74] J.C. Polanco, B.J. Scicluna, A.F. Hill, J. Gotz, Extracellular vesicles isolated from the
brains of rTg4510 mice seed tau protein aggregation in a threshold-dependent
manner, J. Biol. Chem. 291 (2016) 12445–12466.
[75] C. Arenaccio, C. Chiozzini, S. Columba-Cabezas, F. Manfredi, E. Affabris, A. Baur,
M. Federico, Exosomes from human immunodeficiency virus type 1 (HIV-1)-in-
fected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a
Nef- and ADAM17-dependent mechanism, J. Virol. 88 (2014) 11529–11539.
[76] H.C. Christianson, K.J. Svensson, T.H. van Kuppevelt, J.P. Li, M. Belting, Cancer cell
exosomes depend on cell-surface heparan sulfate proteoglycans for their inter-
nalization and functional activity, Proc. Natl. Acad. Sci. U. S. A. 110 (2013)
17380–17385.
[77] S. Tajbakhsh, E. Vivarelli, G. Cuselladeangelis, D. Rocancourt, M. Buckingham,
G. Cossu, A population of myogenic cells derived from the mouse neural-tube,
Neuron 13 (1994) 813–821.
[78] T.J. Smyth, J.S. Redzic, M.W. Graner, T.J. Anchordoquy, Examination of the spe-
cificity of tumor cell derived exosomes with tumor cells in vitro, Biochim. Biophys.
Acta 1838 (2014) 2954–2965.
[79] A. Hoshino, B. Costa-Silva, T.L. Shen, G. Rodrigues, A. Hashimoto, M.T. Mark,
H. Molina, S. Kohsaka, A. Di Giannatale, S. Ceder, S. Singh, C. Williams, N. Soplop,
K. Uryu, L. Pharmer, T. King, L. Bojmar, A.E. Davies, Y. Ararso, T. Zhang, H. Zhang,
J. Hernandez, J.M. Weiss, V.D. Dumont-Cole, K. Kramer, L.H. Wexler,
A. Narendran, G.K. Schwartz, J.H. Healey, P. Sandstrom, K.J. Labori, E.H. Kure,
P.M. Grandgenett, M.A. Hollingsworth, M. de Sousa, S. Kaur, M. Jain, K. Mallya,
S.K. Batra, W.R. Jarnagin, M.S. Brady, O. Fodstad, V. Muller, K. Pantel, A.J. Minn,
M.J. Bissell, B.A. Garcia, Y. Kang, V.K. Rajasekhar, C.M. Ghajar, I. Matei,
H. Peinado, J. Bromberg, D. Lyden, Tumour exosome integrins determine organo-
tropic metastasis, Nature 527 (2015) 329.