ORIGINAL RESEARCH ARTICLE

Distinct RNA profiles in subpopulations

of extracellular vesicles: apoptotic
bodies, microvesicles and exosomes
Rossella Crescitelli1,2, Cecilia Lasser1, Tamas G. Szabo3,
Agnes Kittel4, Maria Eldh1, Irma Dianzani2, Edit I. Buzas3* and
Jan Lotvall1*
1

Department of Internal Medicine and Clinical Nutrition, Krefting Research Centre, University of Gothenburg,
Gothenburg, Sweden; 2Department of Health Sciences, University of Eastern Piedmont, Novara, Italy;
3
Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary; 4Institute of
Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

Introduction: In recent years, there has been an exponential increase in the number of studies aiming
to understand the biology of exosomes, as well as other extracellular vesicles. However, classification
of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion
and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid
cross-contamination.
Method: EVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using
two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000 g, followed by
filtering the supernatant through 0.8 mm pores and pelleting of microvesicles at 12,200 g. In protocol 2,
apoptotic bodies and microvesicles were collected together at 16,500 g, followed by filtering of the
supernatant through 0.2 mm pores and pelleting of exosomes at 120,000 g. Extracellular vesicles were
analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a
Bioanalyzer.
Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller
RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no
RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly
different distribution of size, shape and electron density with typical apoptotic body, microvesicle and
exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the
presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as
these cells do not express CD9.
Conclusions: Our results demonstrate that centrifugation-based protocols are simple and fast systems to
distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and
morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry
analysis.
Keywords: apoptotic bodies; microvesicles; exosomes; extracellular vesicles; ultracentrifugation; characterization; RNA;
electron microscopy

Received: 20 February 2013; Revised: 31 July 2013; Accepted: 16 August 2013; Published: 12 September 2013

xtracellular vesicles (EVs) are membranous vesicles naturally released by most cells (1
9). EVs
can be broadly classified into three main classes,
based primarily on their size and presumed biogenetic
pathways: (a) apoptotic bodies (ABs), 800
5,000 nm
diameter and released by cells undergoing programmed
cell death, (b) microvesicles (MVs), also referred to as

shedding MVs, are large membranous vesicles (50
1,000

nm diameter) that are produced by budding from the
plasma membrane (c) and finally exosomes (EXOs), 40

100 nm diameter vesicles considered to be of endocytic
origin (10,11).
Despite some presumed distinct features, numerous
similarities exist among the different EVs with respect to

Journal of Extracellular Vesicles 2013. # 2013 Rossella Crescitelli et al. This is an Open Access article distributed under the terms of the Creative Commons
Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

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Rossella Crescitelli et al.

their physical characteristics and biochemical composition (12
15), which make the separation of different subsets challenging (12). Because of their small size,
many EVs are below the detection range of conventional
detection methods such as light microscopy. Consequently, recovery and contamination among vesicles
in the separation process cannot be reliably controlled.
Furthermore, isolation protocols and the nomenclature
are not fully standardized in the field at this point.
In most studies, vesicles are isolated by differential
centrifugation steps which are considered to be the
golden standard to isolate different types of EVs
(16). Differential centrifugation involves multiple sequential centrifugations, each time removing the pellet and
the supernatant, and includes increasing the centrifugal
force to isolate smaller and less dense components in
the subsequent steps. In general, centrifugal force at
200
1,500 g are used to pellet cells and cellular
debris, 10,000
20,000 g to pellet vesicles with a size
between 100 and 800 nm (generally called MVs) and
between 100,000 and 200,000g to pellet the smallest
vesicles with a diameter B100 nm (generally referred to
as EXOs) (17).
Besides the size and density of vesicles, the efficiency
to isolate vesicles depends on the shape and viscosity of
the solution, as well as on temperature, centrifugation
time and the type of rotor used for the centrifugation
(fixed-angle rotor or swinging buckets). As vesicles are
heterogeneous, complete separation of vesicles with a
certain diameter and/or density is still unlikely with this
approach. Besides differential centrifugations, filtration
has also been applied to remove larger vesicles from
smaller ones. Although the pore size of filters is often
well defined, increasing forces have to be applied with
decreasing pore size, which can result in artefacts (12,17).
Although flow cytometry and Western blot has been
utilized to identify and characterize nano-sized vesicles
(18), the golden standard remains to be transmission
electron microscopy (TEM) (19), which is the only
method by which both the size and morphology of the
isolated vesicles can be determined simultaneously (12).
Attempts to separate different vesicles to allow analysis
of their diverse functions and description of their different contents also remain crucial for the development of
the field.
In this study, we have used differential centrifugation
steps to achieve a relative separation of ABs, MVs and
EXOs from several different cell lines, with the hypothesis
that the RNA profiles are different in different types of
vesicles, but similar among vesicles from different types
of cells. To do this, three fundamentally different cell lines
were cultured in vitro, including a human mast cell line
(HMC-1), a human erythroleukemia cell line (TF-1)
and a mouse microglia cell line (BV-2). Different EVs
were isolated to determine their respective RNA profiles.

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To determine the morphology of the different vesicles,

the subpopulations of EVs from the different cells were
visualized using TEM of sectioned vesicle pellets.

Materials and methods

Cell cultures
The HMC-1 (J. Butterfield, Mayo Clinic, Rochester, MN,
USA) used in our earlier studies (20,21) was cultured in
IMDM (Sigma-Aldrich, St. Louis, MO, USA) containing 10% foetal bovine serum (FBS, Sigma-Aldrich),
100 U ml 1 penicillin, 100 mg ml 1 streptomycin, 2 mM
L-glutamine and 1.2 mM a-thioglycerol (Sigma-Aldrich).
The cytokine-dependent erythroleukemia cell line TF-1
(ATCC number: CRL-2003) was grown in RPMI 1640
medium supplemented with 10% FBS, 100 U ml 1
penicillin, 100 mg ml 1 streptomycin, 2 mM L-glutamine
(all reagents were from Sigma-Aldrich) and 5 ng ml 1
GM-CSF (granulocyte-macrophage colony-stimulating
factor, Miltenyi Biotec, Lund, Sweden). The BV-2 murine
microglia cells were grown in RPMI supplemented by
10% FBS (Gibco Invitrogen Corporation, Carlsbad, CA,
USA) and 4 mg ml 1 ciprofloxacin (Fresenius Kabi
Deutschland GmbH, Bad Homburg v.d.H, Germany).
For all FBS used in the cell cultures, pure foetal bovine
serum was depleted from EXOs prior to use, by ultracentrifugation at 120,000g for 18 hours, using a Ti45
rotor (Beckman Coulter, Brea, CA, USA). Cell viability
was assessed using trypan blue exclusion methods.
Vesicle isolation
Vesicles were prepared from the supernatant of HMC-1,
TF-1 and BV-2 cells (1
2106 cells ml 1) using two
different centrifugation-based protocols. Briefly, for both
protocols, cells were isolated and removed by pelleting
with centrifugation at 300g for 10 minutes. Vesicles
were then collected from the supernatant through differential centrifugation steps (Fig. 1).
Protocol 1: ABs and MVs were isolated by differential centrifugations and micro-filtration as previously
described (22). The supernatant harvested from the cells
was centrifuged at 2,000 g for 20 minutes to collect ABs.
This supernatant was then filtered by gravity through
0.8 mm filters (GE healthcare, Whatman , UK) to remove
particles 800 nm. The supernatant was again collected
and further used to isolate MVs. MVs were pelleted
by centrifugation at 12,200 g for 40 minutes (Fig. 1A).
All centrifugation steps in both protocols 1 and 2 were
performed at 48C.
Protocol 2: ABsMVs and EXOs were isolated by
differential centrifugations and nano-filtration as previously described (21). ABsMVs were collected together
in this protocol by a centrifugation of the cell supernatant
at 16,500g for 20 minutes. The supernatant from
this step was filtered through 0.2 mm filters (with gentle

Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

RNA in extracellular vesicles

A)

B)
Protocol 1

Protocol 2

300 xg, 10 min

300 xg, 10 min

2,000 xg, 20 min =

Apoptotic bodies (ABs)
0.8 m filter by gravity
12,200 xg, 40 min =
Microvesicles (MVs)

16,500 xg, 20 min =

Apoptotic bodies
+ Microvesicles
(ABs+MVs)
0.2 m filter by pressure
120,000 xg, 70 min =
Exosomes (EXOs)

Fig. 1. Flow chart over two different differential centrifugationbased protocols. Apoptotic bodies (ABs) and microvesicles
(MVs) were isolated separately using protocol 1 (A). ABs and
MVs were isolated together (ABsMVs) followed by exosome
(EXO) isolation, using protocol 2 (B).

pressure) (Sarstedt, Numbrecht-Rommelsdorf, Germany)

to remove particles larger than 200 nm. EXOs were
then pelleted by ultracentrifugation at 120,000 g for
70 minutes (Fig. 1B).

RNA isolation and detection

RNA was isolated from vesicles (n4 for HMC-1 and
TF-1 cells and n3 for BV-2 cells) using miRCURYTM
RNA Isolation Kit (Exiqon, Vedbaek, Denmark) according to the manufacturers protocol. Detection, quality,
yield and size of the vesicular RNA were analyzed using
capillary electrophoresis (Agilent RNA 6000 Nano Kit
on an Agilent 2100 Bioanalyzer, Agilent Technologies,
Santa Clara, CA, USA). One microlitre RNA in solution
was analyzed according to the manufacturers protocol as
previously described (23).
Induction and determination of apoptosis
To induce apoptosis, TF1 cells were incubated with 100 ng
ml 1 of recombinant human TNF-related apoptosisinducing ligand (TRAIL; PeproTech Inc., Rocky Hill,
NJ, USA). Apoptosis was assessed after 2, 4, 8, 24 and
48 hours by using PE Annexin V Apoptosis Detection
Kit I (BD-PharmingenTM, San Jose, CA, USA) according
to the manufacturers protocol. Briefly, after two washes
in cold PBS, cells were resuspended in 1 binding buffer
(10: 0.1 M Hepes/NaOH (pH 7.4) 1.4 M NaCl, 25 mM
CaCl2) at 1106 cells ml 1. One hundred microlitres of
cellular suspension was transferred in a FACS tube and
5 ml of Annexin V-PE antibody and 5 ml of the vital dye
7-Amino-Actinomycin (7-AAD) were added. Cells with
intact membranes exclude 7-AAD, whereas the mem-

brane of necrotic cells is permeable to 7-AAD. Apoptotic

cells are identified by positivity for Annexin-V. After
15 minutes of incubation at room temperature (RT) in
the dark, 400 ml of 1 binding buffer was added and the
fluorescence was determined by a FACSAria (BD Biosciences, San Jose, CA, USA). The flow cytometry data
were analyzed using the FlowJo Software (Tri Star Inc.,
Ashland, OR, USA) (n2). ABs were collected after 4,
24 and 48 hours of TRAIL treatment, while the other
populations of EVs (MVs, ABMVs and EXOs) were
collected after 48 hours only. The RNA profiles were
analyzed in all samples as described above (n2).

Transmission electron microscopy

The vesicular pellets obtained by the two differential
centrifugation-based protocols were submitted to TEM.
Briefly, after isolation (see vesicle isolation section)
pellets were fixed at 48C overnight. The fixative contained
4% paraformaldehyde in 0.01 M phosphate buffer with
pH 7.4 (filtered through 0.22 mm filters). After washing with PBS, the preparations were post-fixed in 1%
OsO4 (Taab Laboratories Equipment Ltd., Aldermaston,
England, UK) for 30 minutes. After rinsing with distilled
water, the pellets were dehydrated in graded ethanol,
including block staining with 1% uranyl-acetate in 50%
ethanol for 30 minutes, and embedded in Taab 812 (Taab).
After overnight polymerization at 608C and sectioning
for TEM, the ultrathin sections were analyzed with a
Hitachi 7100 electron microscope equipped by Megaview
II (lower resolution, Soft Imaging System) digital camera.
Flow cytometry of vesicles
The protein concentration of the vesicle preparations was
measured using the BCATM Protein Assay Kit (Pierce,
Thermo Scientific, Rockford, IL, USA). Antibody-coated
beads were prepared as previously described (20,24).
Briefly, for the immune-isolation, 4-mm-diameter aldehyde/sulfate latex beads (Interfacial Dynamics, Life Technologies, Carlsbad, CA, USA) were incubated with 12.5 mg
purified anti-CD63 antibody (clone H5C6, BD Biosciences), with the same volume of MES buffer under
gentle agitation at RT overnight.
Vesicles (20 mg) were resuspended in PBS and loaded
onto the anti-CD63-coated beads (6 104) and were
incubated overnight at 48C under agitation. Vesicle-coated
beads were incubated for 30 minutes with 100 mM glycine
to block remaining binding sites. The bead
vesicle complexes were washed twice in PBS with 3% FBS (prior
ultracentrifuged at 120,000 g for 18 hours). The bead

vesicle complexes were resuspended in IgG (SigmaAldrich) and incubated for 15 minutes at RT, before being
washed twice more. The tetraspanins CD9, CD63 and
CD81, known to be enriched in EXOs, were investigated
for its presence on the vesicles. The bead
vesicle complexes were incubated with PE-labelled anti-CD9 (clone
M-L13), anti-CD63 (clone H5C6, the same antibody as

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Rossella Crescitelli et al.

used to coat the beads), anti-CD81 (clone JS-81) or the

corresponding isotype control (all antibodies were from
BD Biosciences) for 40 minutes at RT under agitation,
washed twice and then acquired by a FACSAria (BD
Biosciences) (n3). The flow cytometry data were analyzed using the FlowJo Software (Tri Star Inc., Ashland,
OR, USA).

BV-2 cells, but could be observed in MVs from TF-1

cells (Fig. 2B). In the 16,500g pellet, which contained
both ABs and MVs, similar RNA profiles as seen for ABs
were observed in vesicles from all cell lines (Fig. 2C).
In EXOs, the RNA profile from all three cell lines lacked
the rRNA peaks, but showed the presence of small
RNAs (Fig. 2D). Bioanalyzer RNA profiles from the
two different protocols are also illustrated in the same
figure from each cell line (Fig. 2E
G). These data argue
that the rRNA peaks are mainly contributed by the ABs
and not by MVs.
To better understand whether the ABs collected at
2,000 g (protocol 1, Fig. 1) were indeed representative
of cells undergoing programmed cell death, recombinant human TRAIL was used to induce apoptosis in
the TF-1 cells, as TRAIL has previously been shown
to induce apoptosis (25
27). Apoptosis and primary/
secondary necrosis were assessed after 2, 4, 8, 24 and
48 hours by flow cytometry using Annexin V-PE and 7AAD staining, respectively. Upon induction of apoptosis
by TRAIL, a three-fold increase in the proportion of
apoptotic cells was detected at both 2 and 4 hours without

Results
Subpopulations of EVs harbour different RNA
profiles
RNA profiles in the different vesicular fractions from
the three cell lines, HMC-1, TF-1 and BV-2 were analyzed
using a Bioanalyzer. Using the vesicle isolation protocols described in Fig. 1, different RNA profiles were
observed in the vesicular fractions considered to harbour
ABs, MVs and EXOs (Fig. 2A
D). Thus, the technique
reveals two dominant peaks, corresponding to the ribosomal RNA (rRNA) subunits 18S and 28S, in ABs from
HMC-1, TF-1 and BV-2 cells (Fig. 2A). The rRNA peaks
were lacking or were very low in MVs from HMC-1 and
A)
[FU]
50

B)
[FU]

ABs

C)

MVs

40

30
HMC-1

20

10

0
25

[FU]
50

200 500 1000 2000

[nt]

4000

25

[FU]

200 500 1000 2000

[nt]

4000

[FU]
50

40

30
TF-1

20

10

0
25

[nt]

200 500 1000 2000 4000

[FU]

8
6
4
2
0
25

200 500 1000 2000 4000

[nt]

[nt]

200 500 1000 2000 4000

[FU]
5

40

30

20

10

25

[FU]

200 500 1000 2000 4000

[nt]

25

25

20

20

20

15

15

15

10

10

10

10

20

25

[nt]

200 500 1000 2000 4000

25

HMC-1

E)

200 500 1000 2000 4000

200 500 1000 2000 4000

[nt]

25

200 500 1000 2000 4000

Protocol 1 - ABs
40

Protocol 1 - MVs

40
30

Protocol 2 - ABs+MVs

30
20

20

10

10

0
200 500 1000

2000

4000

[nt]

[nt]

[FU]

50

25

[nt]

BV-2

G)

[FU]

[nt]

0
25

TF-1

F)

[FU]
90
80
70
60
50
40
30
20
10
0

[nt]

200 500 1000 2000 4000

200 500 1000 2000 4000

[FU]

25

30

25

0
25

EXOs

[FU]

25

40

BV-2

[FU]

D)

ABs + MVs

[FU]
80
70
60
50
40
30
20
10
0

25

200 500 1000 2000

4000

[nt]

25

200 500 1000 2000

4000

[nt]

Fig. 2. RNA profiles from different subpopulations of extracellular vesicles (EVs). RNA was extracted from vesicles released by
three different cell lines; HMC-1 (human mast cell line), TF-1 (human erythroleukemia cells) and BV-2 (mouse microglia cells).
The electropherograms show the size distribution in nucleotides (nt) and fluorescence intensity (FU) of total RNA in apoptotic bodies
(ABs), microvesicles (MVs), ABs and MVs together (ABsMVs) and exosomes (EXOs). The short peak at 25 nt is an internal
standard. (A) In ABs the most dominant peaks are the 18S and 28S ribosomal RNA. (B) The 18S and 28S rRNA are not evident in
MVs from HMC-1 and BV-2, but only obvious in MVs from TF-1, however at low concentrations. (C) 18S and 28S peaks are evident in
the pellet composed by ABs and MVs together (ABsMVs). (D) In EXOs small RNA is dominating, with no or very small rRNA
peaks detected. (E
G) The overlapping profiles from ABs (in red) and MVs (in blue) and both collected together (ABsMVs  in
green), suggesting that the contribution of 18S and 28S rRNA is by ABs. The electropherograms are representative of n4.

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Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

RNA in extracellular vesicles

any increase in the ratio of necrotic cells (data not

shown). The percentage of apoptotic cells reached 60.2%
by 48 hours. However, from 8 hours there was also an
increase in the ratio of necrotic cells (reaching 9.7 and
22.6% by 24 and 48 hours, respectively). ABs were
collected at 4 hours (when apoptosis was induced without
any necrosis), at 24 hours (when cells showed apoptosis
with moderate ( B10%) necrosis) and at 48 hours (when
cells showed increased necrosis and apoptosis but with
three times as much apoptosis). The effect of apoptosis on
the RNA content in MVs and EXOs were also determined, but due to low-yield MVs and EXOs were only
collected and analyzed at 48 hours. Figure 3 shows RNA
profiles in vesicles collected with/without TRAIL treatment. As expected, after induction of apoptosis by
TRAIL, the amount of rRNA in vesicular fractions
increased, suggesting increased amounts of ABs (Fig.
3A
C). In MVs, prominent rRNA peaks were present
after TRAIL treatment. The increased quantities of
rRNA could also be explained by an increased presence
of ABs in this vesicular fraction (Fig. 3D).
Larger amount of rRNA was observed in the pellet
composed by ABs and MVs after TRAIL treatment
compared to the pellet obtained without TRAIL treatment (Fig. 3E). In the EXO fraction, the RNA profiles were similar under either condition, but a greater
ABs- 4h

A)

[FU]

14

30

12

25

10
8

20

Different morphology of ABs, MVs and EXOs as

visualized by TEM
EVs containing pellets of HMC-1, TF-1 and BV-2 were
visualized by TEM (Fig. 5). Images revealed that the
pellet from the first step of centrifugation using protocol 1 (2,000 g) is composed by elements with chromatin condensation and/or marginalization with the
size range of 800
5,000 nm that are characteristic of
ABs (Fig. 5A1
A3). A very pure pellet was obtained
from the second step (12,200g) after 0.8 mm filtration.
It contained predominantly round and oval shaped,

ABs- 24h

B)

[FU]

quantity of RNAs was evident in the EXO pellet collected

after TRAIL treatment (Fig. 3F).
To verify if the contribution to the rRNA (18S and 28S
subunits) in the ABsMVs pellet was provided by ABs
and not by MVs, a modification of protocol 2 from Fig. 1
was utilized (here termed protocol 2b, see Fig. 4A).
In this protocol, the 2,000 g step was added, aiming
to separate ABs and MVs otherwise collected in the same
pellet in the original protocol 2 (here termed protocol
2a, see Fig. 4A). The comparison of the RNA profiles
showed that the rRNA peaks were most dominant in
ABs and not in MVs from the HMC-1 and TF-1 cell
lines. We can thus conclude that ABs are likely to contribute to a majority of the rRNA present in the pellet
composed by a mixture of ABs and MVs (Fig. 4B).

C)

ABs- 48h

[FU]
150
100

TRAIL

50

+TRAIL

15

6
4

10
5

0
25

200 500 1000 2000 4000

MVs - 48h

D)
[FU]
16
14
12
10

0
25

[nt]

200 500 1000 2000 4000

[nt]

ABs + MVs - 48h

E)
[FU]
300

25

4000

[nt]

EXOs - 48h

F)
[FU]
20

250

200 500 1000 2000

15

200

8
6
4
2
0

150

10

100

50
0

0
25

200 500 1000 2000 4000

[nt]

25

200 500 1000 2000 4000

[nt]

25

200 500 1000 2000 4000

[nt]

Fig. 3. RNA profiles from different subpopulations of extracellular vesicles (EVs) after TRAIL-induced apoptosis. The
electropherograms show the RNA size distribution in nucleotides (nt) and fluorescence intensity (FU) in apoptotic bodies (ABs),
microvesicles (MVs), ABs and MVs together (ABsMVs) and exosomes (EXOs) in TF-1 cells with and without TRAIL treatment. The
short peak at 25 nt is an internal standard. (A
C) RNA profiles from ABs released by TF-1 cells after 4, 24, 48 hours of TRAIL
treatment (in blue) and without TRAIL (in red). After 4 hours (A), 24 hours (B) and 48 hours (C) of TRAIL treatment, in ABs, the
peaks of 18S and 28S rRNAs are more prominent comparing with ABs released in the absence of TRAIL. (D
F) RNA profiles from
MVs, ABsMVs and EXOs released by TF-1 cells after 48 hours of TRAIL treatment (in blue) and without TRAIL (in red).
(D) The low 18S and 28S rRNA peaks in MVs without TRAIL (in red) become much more prominent after TRAIL treatment (in blue).
(E) The highest rRNA peaks are seen in the pellet composed by ABs and MVs together (ABsMVs). (F) After 48 hours of TRAILinduced apoptosis, increased amount of small RNAs is observed in exosomes (EXOs). The electropherograms are representative
of n2.
Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

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Rossella Crescitelli et al.

A)

B)

Protocol 2
Protocol 2a (orginal)
300 xg, 10 min

Protocol 2b (modified)
300 xg, 10 min

2,000 xg, 20 min =

Apoptotic bodies (ABs)

HMC-1

[FU]
14
12
10
8
6
4
2
0

25 200 500 1000 2000 4000

16,500 xg, 20 min =

16,500 xg, 20 min =

ABs+MVs

MVs

[nt]

TF-1

[FU]
50
40
30

0.2 m filter by pressure

0.2 m filter by pressure

20
10
0

120,000 xg, 70 min =

120,000 xg, 70 min =

Exosomes (EXOs)

Exosomes (EXOs)

25

200 500 1000 2000 4000

Protocol 2a - ABs+MVs

[nt]

Protocol 2b - ABs

Protocol 2b - MVs

Fig. 4. Flow chart over the original and modified protocol 2. (A) In the modification of protocol 2, a 2,000g step was added to isolate
apoptotic bodies (ABs) and microvesicles (MVs) separately, prior to EXOs isolation (here called protocol 2b). (B) The RNA profiles
from the different subpopulation of extracellular vesicles (EVs) collected using protocol 2a and 2b. RNA was extracted from vesicles
releases from two different cell lines; HMC-1 and TF-1. Shown here are the overlapping profiles from ABs (ABs
in red), MVs (MVs

in blue) and both of them collected together (ABsMVs  in green), indicating that the contribution of 18S and 28S rRNA is
primarily by ABs. The electopherograms show the size distribution in nucleotides (nt) and fluorescence intensity (FU) of total RNA.
The peak at 25 nt is an internal standard. The electropherograms are representative of n3.

membrane-bound structures of variable size and electron density within the diameter range of 200
800 nm
(Fig. 5B1
B3). As expected, the first pellet obtained
using protocol 2 (16,500 g) was composed by both
ABs and MVs (Fig. 5C1
C3), as it contains elements
with both 800
5,000 nm and 200
800 nm diameter, with
typical characteristics of ABs and MVs. Finally, pellets
from the last step of centrifugation (120,000g) were
composed of 40
100 nm diameter vesicles, consistent
with EXOs (Fig. 5D1
D3).
The electron micrographs thus confirm that the respective pellets contain intact structures primarily within
the expected diameter ranges and with typical morphological characteristics of the ABs, MVs and EXOs
respectively. The images show similar morphology of the
different vesicles from the three different cell lines
analyzed, confirming that they produced similar subpopulation of vesicles isolated by the same centrifugationbased protocols.

CD9, CD63 and CD81 are present on ABs, MVs

and EXOs
The presence of CD63 and CD81 on the surface of
HMC-1 and TF-1 cells was confirmed by flow cytometry,
whereas CD9 was only expressed on the HMC-1 cells,
and not on the TF-1 cells (Fig. 6A).
In order to better characterize the different subpopulation of EVs, a flow cytometry-based evaluation of the
tetraspanins CD9, CD63 and CD81 was performed (24).
To do this, we utilized CD63-antibody-coated beads and
could capture CD63-containing vesicles from all three

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types of vesicles (Fig. 6B) and not exclusively EXOs as

previously suggested (28,29). As expected based on flow
cytometry results from cells, CD63-containing vesicles
that were derived from HMC-1 cells, were positive for all
markers investigated, whereas TF-1-derived vesicles
bound to the CD63-antibody-coated beads expressed
also CD81, as well as CD63, but not CD9 (Fig. 6B).
HMC-1 cells exposed less CD63 than CD81 at the
cell surface (Fig. 6A), whereas conversely there was a
higher level of CD63 than CD81 on the captured vesicles
(Fig. 6B). TF-1 cells, instead, exposed both tetraspanins
at the same level at their surface, and the released vesicles
also expressed these two markers at the same level.

Discussion
In this study, we have applied previously published
centrifugation-based protocols considered appropriate
for the isolation of ABs and MVs, respectively (22).
Furthermore, we used protocols that are considered to
remove ABs and MVs, and to more specifically isolate
EXOs (21). These protocols were utilized to isolate
the different vesicles from the supernatants of cultured
HMC-1, TF-1 and BV-2 cells. Here, we provide evidence
for the presence of clearly different RNA profiles in the
various vesicle fractions, with rRNA being primarily
detectable in ABs, and smaller RNAs without prominent
rRNA peaks in EXOs. The isolates considered to be
MVs contained little or no RNA, except for those from
TF-1 cells. Indeed, electron microscopy of sectioned
pellets of respective vesicle isolation revealed morphology
compatible with predominantly ABs, MVs and EXOs in

Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

RNA in extracellular vesicles

HMC-1

TF-1

BV-2

ABs

MVs

ABs +
MVs

EXOs

Fig. 5. Analysis of ABs, MVs and EXOs by TEM. Micrographs of vesicles released from three different cell lines; HMC-1 (human mast
cell line), TF-1 (human erythroleukemia cells), and BV-2 (mouse microglia cells) are shown. (A1
3) Dense structures show the
chromatin substance in the generally round shaped apoptotic bodies (ABs) with a size of 800
5,000 nm. (B1
3) Microvesicles (MVs) are
diverse in their shape and density, with a size range between 200 and 800 nm. (C1
3) In the pellet obtained by centrifugation at
16,500 g presents the mixture of ABs and MVs. (D1
3) The exosome (EXO) fraction from HMC-1 (D1), TF-1 (D2) and BV-2 (D3)
cells were found to have a diameter of approximately 40
100 nm.

the different fractions, although some contamination

between the fractions cannot be excluded. ABs and
MVs were more diverse in their morphology than the
EXOs. Flow cytometry revealed the presence of CD63 and
CD81 positive vesicles in all fractions from all cell types,
as well as CD9 except in the TF-1-derived vesicles.
For the separate isolation of ABs and MVs, we first
used a 300g centrifugation to remove cells, and a
subsequent centrifugation with 2,000 g to pellet ABs.
Subsequently, the supernatant was filtered and centrifuged at 12,200 g for the pelleting of MVs. Indeed, this
approach separated vesicles containing rRNA, which was
found primarily in the presumed fraction containing ABs,
whereas no or little RNA was found in the MVs fraction,
at least from the HMC-1 and BV-2 cells. The TF-1derived vesicles showed a slightly different RNA profile,
as rRNA profiles also could be seen in the MV fraction.
We do not know at this stage whether the rRNA
identified in the TF-1-derived MVs fractions is indeed
located in MVs, or it is present in other types of vesicles,
or even in protein aggregates co-pelleting with MVs.
However, electron microscopy of the pellets from the

different vesicle isolations shows distinct morphological

differences between ABs and MVs from all cells studied,
which suggests that the rRNA in MVs from TF-1 cells is
not necessarily due to a contamination from ABs, but
may argue that MVs from some cells indeed does contain
RNA.
With an approach previously utilized in our laboratory
(21), a first centrifugation with 16,500g was utilized
after cell removal to collect ABs and MVs together prior
to EXOs isolation. After that, EXOs were isolated by first
passing the supernatant through a 200 nm filter, followed
by 120,000 g ultracentrifugation. Thus, the first pellet
contains both ABs as well as MVs, and the second pellet
only EXOs. Using this approach, the RNA profiles of
the mixed AB/MV pellet were similar to that seen in
ABs using the first protocol, whereas the EXOs contain
primarily small RNA without any prominent rRNA
peaks (21). When a first centrifugation with 2,000 g
was utilized prior to this protocol (to isolate ABs), again
the rRNA profiles were in the HMC-1 cells seen in the
AB fraction, whereas rRNA was found in both the AB
and MV fractions from the TF-1 cell line. It cannot,

Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

(page number not for citation purpose)

Rossella Crescitelli et al.

A)

HMC-1 cells

B)

ABs

TF-1 cells

0 102 103 104 105

MVs

2
3
4
5
0 10 10 10 10

ABs+MVs

EXOs

Isotype
Control
CD9
CD81
CD63

HMC-1

0
0 102

103

104

105

0 102

103

104

105

0
0 102

103

104

105

0 102

103

104

105

0
0 102

103

104

105

0 102

103

104

105

0 102

103

104

105

0 102

103

104

105

TF-1

Fig. 6. Detection and characterization of extracellular vesicles (EVs) by flow cytometry. The CD9, CD63 and CD81 expression on
HMC-1 and TF-1 cells (A) and their expression on different vesicles, using anti-CD63-coated beads, are shown. (B) Cells and vesicles
were immunostained against the tetraspanin (open curve) CD9 (in black), CD63 (in blue) and CD81 (in red) and compared with their
appropriate isotype control (filled curve). The graphs are representative of n3.

however, be excluded that MVs from different cells have

different capacity to carry RNA. Furthermore, it must
also be considered that the RNA content of EVs may
significantly change depending on the state of the cell
(30). After 4 and 24 hours of TRAIL-induced apoptosis, increased concentration of rRNA was observed in
the isolated ABs, compared to ABs from untreated cells.
After 48 hours of TRAIL-induced apoptosis, a greater
concentration of small RNAs was observed also in EXOs.
This is in concordance with previously published data,
showing that p53-induced apoptosis is associated with
increased EXO secretion (31
33).
In many studies, flow cytometry with beads binding
different types of micro- and nano-sized vesicles has been
utilized (24,28,34), for example to determine the presence
of the tetraspanins CD9, CD63 and CD81 on EXOs.
Here, we have utilized beads with anti-CD63 antibodies,
and could find positive signals in all vesicle fractions,
except for CD9 in the TF-1-derived vesicles, which is
not surprising, since TF-1 cells do not express this
tetraspanin.
Comparing two kinds of cell lines analyzed, and the
vesicles from them, HMC-1 cells express less CD63 than
CD81 at the cell surface, whereas conversely there is
lower level of CD81 than CD63 on the captured vesicles.

8
(page number not for citation purpose)

This result could suggest that part of the CD63-vesicles

may be CD81-negative and comes from intracellular
compartments, whereas TF-1 cells expose both tetraspanins at the same level at its surface and the released
vesicles also express these two markers at a similar level.
These data indicate that the levels of CD63 and CD81
must be analyzed both at the surface of vesicle-secreting
cells and in the resulting secreted vesicles. Results
obtained using flow cytometry indicate that this method
is not sufficient to establish that a nano-vesicle fraction
studied is EXOs, and only EXOs. This may suggest
that these surface molecules are not specific for EXOs,
but are also present on ABs ad MVs from both HMC-1
and TF-1 cells. Alternatively, it may suggest that all three
EVs population contain detectable amounts of CD63positive EXOs. Importantly, in all flow cytometry analyses, events were also observed outside of the gates for
the beads (data not shown), suggesting that a portion of
vesicles do not bind to the CD63 beads, regardless of
isolation protocol and vesicle fraction studied, again
supporting the notion that non-CD63-expressing vesicles
are present in all vesicular fractions.
The TEM analysis of the different vesicular fractions
argues strongly that we primarily have ABs, MVs and
EXOs in the different fractions from all the cells studied.

Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

RNA in extracellular vesicles

Indeed, the morphology of all of these vesicles was

characteristically similar to what previously had been
described (22). Importantly, the contamination of, for
example, EXOs in ABs and MVs is possible, but is not
prominent according to the morphological characteristics.
This study shows that ABs, MVs and EXOs contain
fundamentally different RNA profiles, and argues that
MVs isolated from cell cultures often do not contain
considerable amounts of RNA. The rRNA was primarily
found in ABs, which should be considered when the
functionality of RNA in different vesicles is studied.

12.

13.

14.

Acknowledgements

15.

The authors thank Gunnar Nilsson (Karolinska Institute,

Stockholm, Sweden) for the kind gift of the HMC-1 cells. BV-2
cells were kindly provided by Professor Rosario Donato (Perugia,
Italy).

16.

17.

Conflict of interest and funding

This work was funded by grants from the Swedish Research
Council (K2011-56X-20676-04-6) and Krefting Foundation
against Asthma Allergy. R. C. was funded by Istituto
Piemontese per la ricerca sulla Anemia di Diamond-Blackfan, Cariplo and EI.B. by OTKA 84043 and FP7-PEOPLE2011-ITN
PITN-GA-2011-289033 DYNANO.

References
1. Thery C, Ostrowski M, Segura E. Membrane vesicles as
conveyors of immune responses. Nat Rev Immunol. 2009;9:
581
93.
2. Keller S, Sanderson MP, Stoeck A, Altevogt P. Exosomes: from
biogenesis and secretion to biological function. Immunol Lett.
2006;107:102
8.
3. Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular
organelles important in intercellular communication. J Proteomics. 2010;73:1907
20.
4. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek
A, Ratajczak MZ. Membrane-derived microvesicles: important
and underappreciated mediators of cell-to-cell communication.
Leukemia. 2006;20:1487
95.
5. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles:
artefacts no more. Trends Cell Biol. 2009;19:43
51.
6. Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G. The
biogenesis and functions of exosomes. Traffic. 2002;3:321
30.
7. van Niel G, Porto-Carreiro I, Simoes S, Raposo G. Exosomes:
a common pathway for a specialized function. J Biochem.
2006;140:13
21.
8. Johnstone RM. Exosomes biological significance: a concise
review. Blood Cells Mol Dis. 2006;36:315
21.
9. Rajendran L, Honsho M, Zahn TR, Keller P, Geiger KD,
Verkade P, et al. Alzheimers disease beta-amyloid peptides are
released in association with exosomes. Proc Natl Acad Sci
USA. 2006;103:11172
7.
10. Kalra H, Simpson RJ, Ji H, Aikawa E, Altevogt P, Askenase P,
et al. Vesiclepedia: a compendium for extracellular vesicles
with continuous community annotation. PLoS Biol. 2012;10:
e1001450.
11. Simpson RJ, Mathivanan S. Extracellular microvesicles: the
need for internationally recognised nomenclature and stringent

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

purification criteria. J Proteomics Bioinform. 2012;5:ii
ii. doi:

10.4172/jpb.10000e10.
Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, Aradi B,
et al. Membrane vesicles, current state-of-the-art: emerging
role of extracellular vesicles. Cell Mol Life Sci. 2011;68:
2667
88.
Choi DS, Yang JS, Choi EJ, Jang SC, Park S, Kim OY, et al.
The protein interaction network of extracellular vesicles
derived from human colorectal cancer cells. J Proteome Res.
2012;11:1144
51.
de Jong OG, Verhaar MC, Chen Y, Vader P, Gremmels H,
Posthuma G, et al. Cellular stress conditions are reflected in
the protein and RNA content of endothelial cell-derived
exosomes. J Extracell Vesicles. 2012;1:18396, doi: http://dx.
doi.org/10.3402/jev.v1i0.18396
Simpson RJ, Kalra H, Mathivanan S. ExoCarta as a resource
for exosomal research. J Extracell Vesicles. 2012;1:18374, doi:
http://dx.doi.org/10.3402/jev.v1i0.18374
Gould SJ, Raposo G. As we wait: coping with an imperfect
nomenclature for extracellular vesicles. J Extracell Vesicles.
2013;2:20389, doi: http://dx.doi.org/10.3402/jev.v2i0.20389
van der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R.
Classification, functions, and clinical relevance of extracellular
vesicles. Pharmacol Rev. 2012;64:676
705.
Chaput N, Thery C. Exosomes: immune properties and
potential clinical implementations. Semin Immunopathol.
2011;33:419
40.
van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen
TG, Nieuwland R. Optical and non-optical methods for
detection and characterization of microparticles and exosomes.
J Thromb Haemost. 2010;8:2596
607.
Ekstrom K, Valadi H, Sjostrand M, Malmhall C, Bossios A,
Eldh M, et al. Characterization of mRNA and microRNA in
human mast cell-derived exosomes and their transfer to other
mast cells and blood CD34 progenitor cells. J Extracell Vesicles.
2012;1:18389, doi: http://dx.doi.org/10.3402/jev.v1i0.18389
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall
JO. Exosome-mediated transfer of mRNAs and microRNAs is
a novel mechanism of genetic exchange between cells. Nat Cell
Biol. 2007;9:654
9.
Turiak L, Misjak P, Szabo TG, Aradi B, Paloczi K, Ozohanics
O, et al. Proteomic characterization of thymocyte-derived
microvesicles and apoptotic bodies in BALB/c mice. J Proteomics. 2011;74:2025
33.
Lasser C. Identification and analysis of circulating exosomal
microRNA in human body fluids. Methods Mol Biol. 2013;
1024:109
28.
Lasser C, Eldh M, Lotvall J. Isolation and characterization of
RNA-containing exosomes. J Vis Exp. 2012;(59):e3037. doi:
10.3791/3037.
Droin N, Guery L, Benikhlef N, Solary E. Targeting apoptosis
proteins in hematological malignancies. Cancer Lett. 2013;332:
325
34.
Suliman A, Lam A, Srivastava RK. Intracellular mechanisms
of TRAIL: apoptosis through mitochondrial-dependent and
-independent pathways. Oncogene. 2001;20:2122
33.
Berent-Maoz B, Piliponsky AM, Daigle I, Simon HU, LeviSchaffer F. Human mast cells undergo TRAIL-induced
apoptosis. J Immunol. 2013;176:2272
8.
Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie
O, Geuze HJ. Selective enrichment of tetraspan proteins on the
internal vesicles of multivesicular endosomes and on exosomes
secreted by human B-lymphocytes. J Biol Chem. 1998;273:
20121
7.
Bobrie A, Marina Colombo M, Krumeich S, Raposo G, Thery
C. Diverse subpopulations of vesicles secreted by different

Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677

(page number not for citation purpose)

Rossella Crescitelli et al.

30.

31.

32.

33.

intracellular mechanisms are present in exosome preparations

obtained by differential ultracentrifugation. J Extracell Vesicles.
2012;1:18397, doi: http://dx.doi.org/10.3402/jev.v1i0.18397
Tatischeff I, Larquet E, Falcon-Perez JM, Turpin P-Y,
Kruglik SG. Fast characterisation of cell-derived extracellular
vesicles by nanoparticles tracking analysis, cryo-electron
microscopy, and Raman tweezers microspectroscopy. J Extracell Vesicles. 2012;1:19179, doi: http://dx.doi.org/10.3402/jev.
v1i0.19179
Yu X, Harris SL, Levine AJ. The regulation of exosomes
secretion: a novel function of the p53 protein. Cancer Res.
2006;66:4795
801.
Lespagnol A, Duflaut D, Beekman C, Blanc L, Fiucci G,
Marine JC, et al. Exosome secretion, including the DNA
damage-induced p53-dependent secretory pathway, is severely
compromised in TSAP6/Steap3-null mice. Cell Death Differ.
2008;15:1723
33.
Lehmann BD, Paine MS, Brooks AM, McCubrey JA, Renegar
RH, Wang R, et al. Senescence-associated exosome release

10
(page number not for citation purpose)

from human prostate cancer cells. Cancer Res. 2008;68:

7864
71.
34. Freyssinet JM, Toti F. Membrane microparticle determination:
at least seeing whats being sized! J Thromb Haemost. 2010;
8:311
4.
*Edit I. Buzas
Department of Genetics, Cell and Immunobiology
Semmelweis University
Budapest, Nagyvarad ter 4,
1089 Hungary
Email: edit.buzas@gmail.com
*Jan Lotvall
Krefting Research Centre
University of Gothenburg
BOX 424, SE405 30 Goteborg
Sweden
Email: jan.lotvall@gu.se

Citation: Journal of Extracellular Vesicles 2013, 2: 20677 - http://dx.doi.org/10.3402/jev.v2i0.20677