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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Experiment number
  • Experiments differ in Isolation method
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  • Experiments differ in Vesicle type
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  • Experiments differ in Sample type/Isolation method
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  • Experiments differ in Sample type, Vesicle type
Experiment number
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  • Experiments differ in Vesicle type
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Experiment number
  • Experiments differ in Sample type/Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV150108 1/4 Homo sapiens MCF-7 (d)(U)C
Filtration
Tosar JP 2015 67%

Study summary

Full title
All authors
Tosar JP, Gámbaro F, Sanguinetti J, Bonilla B, Witwer KW, Cayota A.
Journal
Nucleic Acids Res
Abstract
Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulat (show more...)Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19-60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5' tRNA halves and 5' RNA Y4-derived fragments of 31-33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF-7
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
150
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
7
Wash: time (min)
150
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing;ddPCR
Database
Yes
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
64
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
4U/mL
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Not Reported
NTA
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV150009 1/1 Homo sapiens Placental perfusate (d)(U)C
Filtration
Dragovic RA 2015 67%

Study summary

Full title
All authors
Dragovic RA, Collett GP, Hole P, Ferguson DJ, Redman CW, Sargent IL, Tannetta DS
Journal
Methods
Abstract
The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesi (show more...)The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ?300nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ?50nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of standard 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies. (hide)
EV-METRIC
67% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Placental perfusate
Sample origin
NAY
Focus vesicles
exosomes / microvesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Adj. k-factor
162 (pelleting)
Protein markers
EV: Alix/ CD63
non-EV: CD45/ CD235a/b/ CD41
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Placental perfusate
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TST28.39
Pelleting: adjusted k-factor
162.0
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63
Detected contaminants
CD41/ CD235a/b/ CD45
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV150004 1/2 Homo sapiens NAY (d)(U)C
DG
Clark DJ 2015 67%

Study summary

Full title
All authors
Clark DJ, Fondrie WE, Liao Z, Hanson PI, Fulton A, Mao L, Yang AJ
Journal
Anal Chem
Abstract
Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into t (show more...)Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as true exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Protein markers
EV: Alix/ TSG101/ CD63
non-EV: Beta-actin/ Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.14-1.19
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ TSG101
Detected contaminants
Cell organelle protein/ Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV150008 1/1 Homo sapiens NAY (d)(U)C
DG
Filtration
Chan YK 2015 67%

Study summary

Full title
All authors
Chan YK, Zhang H, Liu P, Tsao SW, Lung ML, Mak NK, Ngok-Shun Wong R, Ying-Kit Yue P
Journal
Int J Cancer
Abstract
Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling (show more...)Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 ?g/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (? 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: CD63/ CD9
non-EV: Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.17-1.19
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.2 M
Highest density fraction
2.5 M
Orientation
Top-down
Rotor type
90Ti
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV230654 1/1 Mycobacterium tuberculosis H37Rv (d)(U)C
DC
DG
Filtration
UF
Lee J 2015 63%

Study summary

Full title
All authors
Lee J, Kim SH, Choi DS, Lee JS, Kim DK, Go G, Park SM, Kim SH, Shin JH, Chang CL, Gho YS
Journal
Proteomics
Abstract
The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exo (show more...)The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160). (hide)
EV-METRIC
63% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density cushion
Density gradient
Filtration
Ultrafiltration
Protein markers
EV: LprG
non-EV: None
Proteomics
yes
EV density (g/ml)
1,17
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mycobacterium tuberculosis
Sample Type
Cell culture supernatant
EV-producing cells
H37Rv
EV-harvesting Medium
serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
10,3
Sample volume (mL)
4,8
Orientation
Bottom-up
Speed (g)
200000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Density cushion
Density medium
Sucrose
Cushion volume
1,5
Density of the cushion
0.8-2.0 M
Centrifugation time
120
Centrifugation speed
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
LprG
Proteomics database
ProteomeXchange
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
40-50
EV concentration
Yes
Particle yield
Total particle count
EM
EM-type
Transmission­-EM
Image type
Wide-field
Report size (nm)
20-250
EV230660 1/1 Pseudomonas syringae Lz4W (d)(U)C
DG
Filtration
Kulkarni HM 2015 57%

Study summary

Full title
All authors
Kulkarni HM, Swamy ChV, Jagannadham MV
Journal
Data Brief
Abstract
Outer membrane vesicles (OMVs) of gram-negative bacteria are released during all growth phases and p (show more...)Outer membrane vesicles (OMVs) of gram-negative bacteria are released during all growth phases and play an important role in bacterial physiology. They consist of lipids, proteins, lipopolysaccharides and other molecules. The OMVs of the Antarctic bacterium Pseudomonas syringae Lz 4W were isolated and identified their proteins. The mass spectral data set deposited with PRIDE, accession number PXD 000221 is presented in this report. The proteins identified from the OMVs of P. syringae Lz4W, data of this study were published in the Journal of proteome research [1]. (hide)
EV-METRIC
57% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Pseudomonas syringae
Sample Type
Cell culture supernatant
EV-producing cells
Lz4W
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
150264
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
20%
Highest density fraction
70%
Orientation
Top­-down
Speed (g)
164609
Duration (min)
360
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: speed (g)
163202
Pelleting: adjusted k-factor
7,59E
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per million cells
Proteomics database
ProteomeXchange
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
60-80
EM
EM-type
Transmission­-EM
Image type
Wide-field
Report size (nm)
60-100
EV150034 2/2 Homo sapiens Blood plasma (d)(U)C
SEC
de Menezes-Neto A 2015 57%

Study summary

Full title
All authors
de Menezes-Neto A, Sáez MJ, Lozano-Ramos I, Segui-Barber J, Martin-Jaular L, Ullate JM, Fernandez-Becerra C, Borrás FE, Del Portillo HA
Journal
J Extracell Vesicles
Abstract
Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major cha (show more...)Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of classical exosome markers. Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS. (hide)
EV-METRIC
57% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
NAY
Focus vesicles
Vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC
Protein markers
EV: CD81/ GAPDH/ CD9
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
GAPDH
ELISA
Detected EV-associated proteins
GAPDH
Characterization: Particle analysis
NTA
EM
EM-type
immune EM/ cryo EM
EM protein
CD5L
Image type
Close-up
EV230672 1/4 Vibrio cholerae A1552 (d)(U)C
DG
Filtration
Sjöström AE 2015 56%

Study summary

Full title
All authors
Sjöström AE, Sandblad L, Uhlin BE, Wai SN
Journal
Sci Rep
Abstract
Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Outer membrane vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
0 (pelleting)
Protein markers
EV: OmpU
non-EV: RpoS
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Vibrio cholerae
Sample Type
Cell culture supernatant
EV-producing cells
A1552
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
0
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
7
Lowest density fraction
10%
Highest density fraction
45%
Orientation
Top­-down
Speed (g)
180000
Duration (min)
120
Fraction volume (mL)
0.1-0.3
Fraction processing
Centrifugation
Pelleting: volume per fraction
0.1-0.3
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
1,70E
Pelleting-wash: volume per pellet (mL)
60
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
OmpU
Not detected contaminants
RpoS
Characterization: RNA analysis
RNA analysis
Type
RNA ­sequencing/ Northern blot
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission­-EM/ cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
25-200
EV230657 1/2 Escherichia coli O104:H4 (d)(U)C
DG
Filtration
Kunsmann L 2015 56%

Study summary

Full title
All authors
Kunsmann L, Rüter C, Bauwens A, Greune L, Glüder M, Kemper B, Fruth A, Wai SN, He X, Lloubes R, Schmidt MA, Dobrindt U, Mellmann A, Karch H, Bielaszewska M
Journal
Sci Rep
Abstract
The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and ha (show more...)The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
LB226692
Focus vesicles
Outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: OmpA/ ShET1/ Flagellin/ Stx2a
non-EV: Pic/ SigA/ SepA
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
O104:H4
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
2
Orientation
Bottom-up
Speed (g)
288000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
OmpA/ ShET1/ Flagellin/ Stx2a
Not detected contaminants
Pic/ SigA/ SepA
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
EM
EM-type
Immuno-­EM
Image type
Close-up
EV230629 2/9 Haemophilus influenzae Rd KW20 (d)(U)C
DG
Filtration
Roier S 2015 56%

Study summary

Full title
All authors
Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S
Journal
Int J Med Microbiol
Abstract
Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Pure outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: RpoA
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development
Sample
Species
Haemophilus influenzae
Sample Type
Cell culture supernatant
EV-producing cells
Rd KW20
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
240
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
144000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Protein Yield (µg)
per mL/OD490
Western Blot
Not detected contaminants
RpoA
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV230629 4/9 Haemophilus influenzae Hib strain Eagan (d)(U)C
DG
Filtration
Roier S 2015 56%

Study summary

Full title
All authors
Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S
Journal
Int J Med Microbiol
Abstract
Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Pure outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: RpoA
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development
Sample
Species
Haemophilus influenzae
Sample Type
Cell culture supernatant
EV-producing cells
Hib strain Eagan
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
240
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
144000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Protein Yield (µg)
per mL/OD490
Western Blot
Not detected contaminants
RpoA
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV230629 6/9 Haemophilus influenzae NTHi 2019-R (d)(U)C
DG
Filtration
Roier S 2015 56%

Study summary

Full title
All authors
Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S
Journal
Int J Med Microbiol
Abstract
Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Pure outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: RpoA
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development
Sample
Species
Haemophilus influenzae
Sample Type
Cell culture supernatant
EV-producing cells
NTHi 2019-R
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
240
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
144000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Protein Yield (µg)
per mL/OD490
Western Blot
Not detected contaminants
RpoA
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV140127 2/5 Homo sapiens Du145 (d)(U)C
DG
Webber JP 2015 56%

Study summary

Full title
All authors
Webber JP, Spary LK, Sanders AJ, Chowdhury R, Jiang WG, Steadman R, Wymant J, Jones AT, Kynaston H, Mason MD, Tabi Z, Clayton A
Journal
Oncogene
Abstract
Activation of myofibroblast rich stroma is a rate-limiting step essential for cancer progression. Th (show more...)Activation of myofibroblast rich stroma is a rate-limiting step essential for cancer progression. The responsible factors are not fully understood, but TGFβ1 is probably critical. A proportion of TGFβ1 is associated with extracellular nano-vesicles termed exosomes, secreted by carcinoma cells, and the relative importance of soluble and vesicular TGFβ in stromal activation is presented. Prostate cancer exosomes triggered TGFβ1-dependent fibroblast differentiation, to a distinctive myofibroblast phenotype resembling stromal cells isolated from cancerous prostate tissue/ supporting angiogenesis in vitro and accelerating tumour growth in vivo. Myofibroblasts generated using soluble TGFβ1 were not pro-angiogenic or tumour-promoting. Cleaving heparan sulphate side chains from the exosome surface had no impact on TGFβ levels yet attenuated SMAD-dependent signalling and myofibroblastic differentiation. Eliminating exosomes from the cancer cell secretome, targeting Rab27a, abolished differentiation and lead to failure in stroma-assisted tumour growth in vivo. Exosomal TGFβ1 is therefore required for the formation of tumour-promoting stroma. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: TSG101/ CD63/ TGF-beta1/ CD81/ MHC class-I/ Alix/ 5T4/ CD9
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Du145
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Density gradient
Type
Not specified
Number of initial discontinuous layers
Not specified
Lowest density fraction
Not specified
Highest density fraction
Not specified
Total gradient volume, incl. sample (mL)
Not specified
Sample volume (mL)
Not spec
Orientation
Not specified
Rotor type
Not specified
Speed (g)
Not specified
Duration (min)
Not specified
Fraction volume (mL)
Not specified
Fraction processing
Not specified
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ 5T4/ MHC class-I/ TSG101
Detected contaminants
Calnexin
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9/ TGF-beta1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
115
EM
EM-type
Transmission-EM
Image type
Close-up
EV150106 1/1 Homo sapiens HMEC-1 DG
(d)(U)C
van Balkom BW 2015 56%

Study summary

Full title
All authors
van Balkom BW, Eisele AS, Pegtel DM, Bervoets S, Verhaar MC.
Journal
J Extracell Vesicles
Abstract
Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and (show more...)Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a small RNA-dependent manner. Recent deep sequencing studies in exosomes from lymphocytic origin revealed a broad spectrum of small RNAs. However, selective depletion or incorporation of small RNA species into endothelial exosomes has not been studied extensively. With next generation sequencing, we identified all known non-coding RNA classes, including microRNAs (miRNAs), small nucleolar RNAs, yRNAs, vault RNAs, 5p and 3p fragments of miRNAs and miRNA-like fragments. In addition, we mapped many fragments of messenger RNAs (mRNAs) and mitochondrial RNAs (mtRNAs). The distribution of small RNAs in exosomes revealed a considerable overlap with the distribution in the producing cells. However, we identified a remarkable enrichment of yRNA fragments and mRNA degradation products in exosomes consistent with yRNAs having a role in degradation of structured and misfolded RNAs in close proximity to endosomes. We propose that endothelial endosomes selectively sequester cytoplasmic RNA-degrading machineries taking part in gene regulation. The release of these regulatory RNAs via exosomes may have implications for endothelial cell-cell communication. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: GAPDH/ Flotillin1/ CD9/
non-EV: Histone H2A.X/ Lamin A / C/ ATP5A/ Tom20
Proteomics
no
EV density (g/ml)
1.07 - 1.12
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HMEC-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
1h at 200000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: time (min)
60
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0.25M
Highest density fraction
2.0M
Sample volume (mL)
0.25
Orientation
Bottom-up
Rotor type
Not specified
Speed (g)
190000
Duration (min)
960
Fraction volume (mL)
0.4
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
GAPDH/ Flotillin1/ CD9
Not detected contaminants
Lamin A/C/ Histone H2A.X/ ATP5A/ Tom20
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNAsequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
EM
EM-type
Transmission-EM
Image type
Wide-field
EV150003 1/2 Homo sapiens NAY (d)(U)C Saari H 2015 56%

Study summary

Full title
All authors
Saari H, Lázaro-Ibáñez E, Viitala T, Vuorimaa-Laukkanen E, Siljander P, Yliperttula M
Journal
J Control Release
Abstract
BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate int (show more...)BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. METHODS: EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. RESULTS: Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. CONCLUSIONS: Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes / microvesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
52.47 (washing)
Protein markers
EV: CD81/ Alpha-tubulin/ TSG101/ CD63/ CD9
non-EV: GAPDH
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Wash: volume per pellet (ml)
1
Wash: Rotor Type
TLA55
Wash: adjusted k-factor
52.47
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin
Detected contaminants
GAPDH
ELISA
Detected EV-associated proteins
Alpha-tubulin
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ cryo EM
Image type
Close-up
EV150016 2/2 Homo sapiens Ascites (d)(U)C
DC
Filtration
Pospichalova V 2015 56%

Study summary

Full title
All authors
Pospichalova V, Svoboda J, Dave Z, Kotrbova A, Kaiser K, Klemova D, Ilkovics L, Hampl A, Crha I, Jandakova E, Minar L, Weinberger V, Bryja V
Journal
J Extracell Vesicles
Abstract
Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and quali (show more...)Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization. (hide)
EV-METRIC
56% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Ascites
Sample origin
NAY
Focus vesicles
exosomes / microvesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DC
Filtration
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: Alix/ HSP70/ TSG101/ CD63
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Ascites
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
190
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
253.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
EM protein
CD63
Image type
Close-up, Wide-field
EV150002 1/1 Homo sapiens NAY (d)(U)C
DG
Phinney DG 2015 56%

Study summary

Full title
All authors
Phinney DG, Di Giuseppe M, Njah J, Sala E, Shiva S, St Croix CM, Stolz DB, Watkins SC, Di YP, Leikauf GD, Kolls J, Riches DW, Deiuliis G, Kaminski N, Boregowda SV, McKenna DH, Ortiz LA
Journal
Nat Commun
Abstract
Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and (show more...)Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Protein markers
EV: CD63/ CD9/ MFGE8
non-EV:
Proteomics
no
EV density (g/ml)
1.09-1.14
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ MFGE8
ELISA
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV150027 2/2 Homo sapiens NAY (d)(U)C
DG
Filtration
Kharaziha P 2015 56%

Study summary

Full title
All authors
Kharaziha P, Chioureas D, Rutishauser D, Baltatzis G, Lennartsson L, Fonseca P, Azimi A, Hultenby K, Zubarev R, Ullén A, Yachnin J, Nilsson S, Panaretakis T
Journal
Oncotarget
Abstract
Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) whi (show more...)Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ CD82/ Alix/ Rab5/ Caveolin1/ CD9
non-EV: Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.12-1.19
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.2
Highest density fraction
2
Orientation
Top-down
Speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD81/ CD9/ TSG101/ CD82/ Rab5/ Caveolin1
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
CD82/ Rab5/ Caveolin1
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up
EV150010 1/1 Homo sapiens NAY (d)(U)C
DG
Keerthikumar S 2015 56%

Study summary

Full title
All authors
Keerthikumar S, Gangoda L, Liem M, Fonseka P, Atukorala I, Ozcitti C, Mechler A, Adda CG, Ang CS, Mathivanan S
Journal
Oncotarget
Abstract
Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocyti (show more...)Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes / Ectosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Adj. k-factor
276.6 (pelleting)
Protein markers
EV: Alix/ CD81/ TSG101/ CD63
non-EV: MMP2/ Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.100
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
1060
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
276.6
Density gradient
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Pelleting-wash: volume per pellet (mL)
1
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD81/ TSG101
Detected contaminants
Cell organelle protein/ MMP2
Characterization: Particle analysis
EM
EM-type
transmission EM/ atomic force EM
Image type
Wide-field
EV150021 1/1 Homo sapiens NAY (d)(U)C
DG
Duchez AC 2015 56%

Study summary

Full title
All authors
Duchez AC, Boudreau LH, Bollinger J, Belleannée C, Cloutier N, Laffont B, Mendoza-Villarroel RE, Lévesque T, Rollet-Labelle E, Rousseau M, Allaeys I, Tremblay JJ, Poubelle PE, Lambeau G, Pouliot M, Provost P, Soulet D, Gelb MH, Boilard E
Journal
Proc Natl Acad Sci U S A
Abstract
Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) cal (show more...)Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
microparticles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Protein markers
EV: TSG101/ VDAC
non-EV:
Proteomics
no
EV density (g/ml)
1.1-1.14
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101/ VDAC
ELISA
Detected EV-associated proteins
VDAC
Characterization: Particle analysis
DLS
EV150103 7/7 Homo sapiens HUVEC (d)(U)C Dieudé M 2015 55%

Study summary

Full title
All authors
Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ
Journal
J Transl Med
Abstract
Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide)
EV-METRIC
55% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Apoptosis
Focus vesicles
apoptotic exosome-like vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
127.9 (pelleting)
Protein markers
EV: LG3/ Fibronectin/ proteasome-alpha3/ Syntenin/ TCTP
non-EV: Tubulin/ GM130
Proteomics
yes
Show all info
Study aim
Function, Biomarker, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
Serum free medium
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
1080
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
127.9
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
300
Western Blot
Detected EV-associated proteins
Syntenin, Fibronectin, TCTP, proteasome-alpha3, LG3
Not detected contaminants
GM130, Tubulin
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BDCantoII Special Order Research Product
Hardware adjustment
This high sensitivity Flow cytometer (hsFCM) is equipped with a small particle option. The forward scatter (FSC) on this dedicated equipment is coupled to a photomultiplier tube (PMT) with a 488 nm solid state;100mW output blue laser (rather than the conventional 20 mW);and includes a 633nmHeNe;20mW output red laser and a 405 nm solid state diode;50mW output violet laser. The hsFCM includes a FSC-PMT and a Fourier optical transformation unit;which reduces the background noise and increases the angle of diffusion;therby enhancing the detection of small-diameter particles.
Calibration bead size
0.09,0.45,0.84,1,3.2
Report type
Median
Reported size (nm)
100-200
EV concentration
Yes
Particle yield
3.50E+07 particles/million cells
EM
EM-type
Transmission-EM/ Immune-EM
EM protein
LG3;proteasome-alpha3
Image type
Close-up, Wide-field
EV150007 1/10 Homo sapiens NAY (d)(U)C
UF
qEV
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
UF
qEV
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 4/10 Homo sapiens NAY (d)(U)C
Filtration
UF
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
UF
Protein markers
EV: TSG101/ HSP70/ Flotilin1/ CD63
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 5/10 Homo sapiens NAY (d)(U)C
DG
Filtration
UF
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
UF
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Rotor type
50.2Ti
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EV150007 7/10 Homo sapiens NAY (d)(U)C
Exo-spin
Filtration
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Exo-spin
Filtration
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Commercial kit
Exo-spin
Other
Name other separation method
Exo-spin
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV150007 8/10 Homo sapiens NAY (d)(U)C
ExoQuick
Filtration
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Filtration
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV140124 2/6 Homo sapiens LNCaP ExoQuick Ramteke, Anand 2015 50%

Study summary

Full title
All authors
Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep
Journal
Mol Carcinog
Abstract
Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: CD63/ MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ HSP90/ Annexin II/ alpha-tubulin/ / Alix/ CD81/ HSP70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
LNCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial
Separation Method
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Lowry­-based assay
Western Blot
Detected EV-associated proteins
CD63/ HSP90/ Annexin II/ alpha-tubulin/ / MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ HSP70/ Alix/ CD81
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
187
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 0.26E8
EM
EM-type
Transmission-EM
Image type
Close-up
EV140124 4/6 Homo sapiens LNCaP ExoQuick Ramteke, Anand 2015 50%

Study summary

Full title
All authors
Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep
Journal
Mol Carcinog
Abstract
Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide)
EV-METRIC
50% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Hypoxia
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: CD63/ MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ HSP90/ Annexin II/ alpha-tubulin/ / Alix/ CD81/ HSP70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
LNCaP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial
Separation Method
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Lowry­-based assay
Western Blot
Detected EV-associated proteins
Alix/ Annexin II/ alpha-tubulin/ / MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ CD63/ HSP90/ HSP70/ CD81
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
141
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 0.24E8
EM
EM-type
Transmission-EM
Image type
Close-up
EV230662 5/6 Mycobacterium smegmatis mc²155 (d)(U)C
Filtration
Kumar S 2015 45%

Study summary

Full title
All authors
Kumar S, Mittal E, Deore S, Kumar A, Rahman A, Krishnasastry MV
Journal
Front Cell Infect Microbiol
Abstract
The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was r (show more...)The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms. (hide)
EV-METRIC
45% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
pTlyA transformed
Focus vesicles
Membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TlyA/ DnaK
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Mycobacterium smegmatis
Sample Type
Cell culture supernatant
EV-producing cells
mc²155
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-100
Pelleting: speed (g)
150000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
TlyA/ DnaK
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission­-EM/ immuno-EM
EM protein
TlyA
Image type
Close-up
EV230657 2/2 Escherichia coli O104:H4 (d)(U)C
DG
Filtration
Kunsmann L 2015 45%

Study summary

Full title
All authors
Kunsmann L, Rüter C, Bauwens A, Greune L, Glüder M, Kemper B, Fruth A, Wai SN, He X, Lloubes R, Schmidt MA, Dobrindt U, Mellmann A, Karch H, Bielaszewska M
Journal
Sci Rep
Abstract
The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and ha (show more...)The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells. (hide)
EV-METRIC
45% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
C227-11Φcu
Focus vesicles
Outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: OmpA/ ShET1/ Flagellin
non-EV: Pic/ SigA/ SepA
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
O104:H4
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
2
Orientation
Bottom-up
Speed (g)
288000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
OmpA/ ShET1/ Flagellin
Not detected contaminants
Pic/ SigA/ SepA
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV230510 2/4 Mus musculus RAW264.7 (d)(U)C
DG
Athman JJ 2015 44%

Study summary

Full title
All authors
Athman JJ, Wang Y, McDonald DJ, Boom WH, Harding CV, Wearsch PA
Journal
J Immunol
Abstract
Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases m (show more...)Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Live mycobacterium tubercolusis
Focus vesicles
extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: CD9/ CD63/ LpqH/ LAM/ LM/ MHCII/ Mtb
non-EV: None
Proteomics
no
EV density (g/ml)
1.11-1.17
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
RAW264.7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: time (min)
70
Wash: Rotor Type
TLA-100.3
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0.2 M
Highest density fraction
1.8 M
Total gradient volume, incl. sample (mL)
11.7
Sample volume (mL)
0.2
Orientation
Top-down
Speed (g)
110000
Duration (min)
1140
Fraction volume (mL)
1
Fraction processing
Precipitation of all proteins/vesicles (e.g. through acidification, Amphipol,...)
Other
Name other separation method
Density gradient
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ LpqH/ LAM/ LM
Other 1
Immunofluorescence
Detected EV-associated proteins
CD9/ CD63/ Mtb
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
95-105
EM
EM-type
Transmission-EM/ Immuno-EM
EM protein
CD9/ MHCII/ Mtb
Image type
Close-up
EV210504 1/5 Macaca mulatta Brain tissue (d)(U)C
DG
Yelamanchili SV 2015 44%

Study summary

Full title
All authors
Yelamanchili SV, Lamberty BG, Rennard DA, Morsey BM, Hochfelder CG, Meays BM, Levy E, Fox HS
Journal
PLoS Pathog
Abstract
Recent studies have found that extracellular vesicles (EVs) play an important role in normal and dis (show more...)Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders. (hide)
EV-METRIC
44% (51st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Brain tissue
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: TSG101/ CD63/ CD81/ SNAP 25/ HSP70/ Synaptophysin/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Macaca mulatta
Sample Type
Brain tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
37
Wash: time (min)
60
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0.25M
Highest density fraction
2.0M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
2
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
6
Fraction processing
Centrifugation
Pelleting: duration (min)
60
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ HSP70/ CD81
Not detected EV-associated proteins
Synaptophysin/ SNAP 25
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ RNA sequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up
EV210395 1/1 Homo sapiens Bone-marrow derived mesenchymal stem cells dUC Shabbir A 2015 44%

Study summary

Full title
All authors
Shabbir A, Cox A, Rodriguez-Menocal L, Salgado M, Van Badiavas E
Journal
Stem Cells Dev
Abstract
Although chronic wounds are common and continue to be a major cause of morbidity and mortality, trea (show more...)Although chronic wounds are common and continue to be a major cause of morbidity and mortality, treatments for these conditions are lacking and often ineffective. A large body of evidence exists demonstrating the therapeutic potential of mesenchymal stem cells (MSCs) for repair and regeneration of damaged tissue, including acceleration of cutaneous wound healing. However, the exact mechanisms of wound healing mediated by MSCs are unclear. In this study, we examined the role of MSC exosomes in wound healing. We found that MSC exosomes ranged from 30 to 100-nm in diameter and internalization of MSC exosomes resulted in a dose-dependent enhancement of proliferation and migration of fibroblasts derived from normal donors and chronic wound patients. Uptake of MSC exosomes by human umbilical vein endothelial cells also resulted in dose-dependent increases of tube formation by endothelial cells. MSC exosomes were found to activate several signaling pathways important in wound healing (Akt, ERK, and STAT3) and induce the expression of a number of growth factors [hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), nerve growth factor (NGF), and stromal-derived growth factor-1 (SDF1)]. These findings represent a promising opportunity to gain insight into how MSCs may mediate wound healing. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
dUC
Protein markers
EV: Flotillin1/ CD9/ CD63/ Beta-actin/ GAPDH/ phosphoStat3 (Tyr750)/ total Stat3/ phospho AKT (Ser473)/ total AKT/ phospho ERK 1/2 (Thr202/204)/ total ERK 1/2/ TSG101/ HSP70/ Alix/ CD81
non-EV: GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Bone-marrow derived mesenchymal stem cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
3h at 100,000g/ Other preparation
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Other
Name other separation method
dUC
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ Beta-actin/ GAPDH/ phosphoStat3 (Tyr750)/ total Stat3/ phospho AKT (Ser473)/ total AKT/ phospho ERK 1/2 (Thr202/204)/ total ERK
Not detected contaminants
GM130
Characterization: Lipid analysis
No
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
30-100
EV210169 1/1 Homo sapiens Blood plasma (d)(U)C
IAF
Filtration
DG
Guescini, Michele 2015 44%

Study summary

Full title
All authors
Michele Guescini, Barbara Canonico, Francesco Lucertini, Serena Maggio, Giosué Annibalini, Elena Barbieri, Francesca Luchetti, Stefano Papa, Vilberto Stocchi
Journal
PLoS One
Abstract
In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble (show more...)In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and alpha-sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1-5% of the total and that 60-65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis. (hide)
EV-METRIC
44% (76th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Immunoaffinity capture (non-commercial)
Filtration
DG
Protein markers
EV: CD81/ TSG101/ Alpha-sarcoglycan
non-EV: None
Proteomics
no
EV density (g/ml)
1.11
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
12.5
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
Not specified
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
60
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Immunoaffinity capture
Selected surface protein(s)
Alpha-sarcoglycan
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Alpha-sarcoglycan/ TSG101
Flow cytometry
Type of Flow cytometry
Not specified
Calibration bead size
1
Detected EV-associated proteins
CD81/ Alpha-sarcoglycan
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
EV210117 1/3 Homo sapiens Kelly (d)(U)C Helge Haug, Bjørn 2015 44%

Study summary

Full title
All authors
Bjørn Helge Haug, Øyvind H Hald, Peter Utnes, Sarah A Roth, Cecilie Løkke, Trond Flægstad, Christer Einvik
Journal
Anticancer Res
Abstract
Background: In recent years, evidence has accumulated indicating that both normal and cancer cells c (show more...)Background: In recent years, evidence has accumulated indicating that both normal and cancer cells communicate via the release and delivery of macromolecules packed into extracellular membrane vesicles. Materials and methods: We isolated nano-sized extracellular vesicles from MYCN-amplified neuroblastoma cell lines using ultracentrifugation and exosome precipitation (Exoquick) protocols. These vesicles were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis and western blotting. Exosomal miRNA profiles were obtained using a reverse transcription-polymerase chain reaction (RT-PCR) ready-to-use panel measuring a total of 742 miRNAs. Results: In this study, we showed that MYCN-amplified neuroblastoma cell lines secrete populations of miRNAs inside small exosome-like vesicular particles. These particles were shown to be taken-up by recipient cells. By profiling the miRNA content, we demonstrated high expression of a group of established oncomirs in exosomes from two MYCN-amplified neuroblastoma cell lines. Despite the fact that other studies have demonstrated the ability of exosomal miRNAs both to repress mRNA targets and to stimulate Toll-like receptor-8 (TLR8) signaling in recipient cells, we did not observe these effects with exosomes from MYCN-amplified neuroblastoma cells. However, functional enrichment analysis reveals that mRNA targets of highly expressed exosomal miRNAs are associated with a range of cellular and molecular functions related to cell growth and cell death. Conclusion: MYCN-amplified neuroblastoma cell lines secrete exosome-like particles containing oncogenic miRNAs. This work showed for the first time that neuroblastoma cells secrete exosome-like particles containing miRNAs with potential roles in cancer progression. These findings indicate a new way for MYCN-amplified neuroblastoma cells to interact with the tumor environment. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: actin/ N-myc/ GRP78
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Kelly
EV-harvesting Medium
EV-depleted medium;Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Preparation of EDS
overnight (16h) at >=100,000g + filtration 0.2 µm
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
110 000
Characterization: Protein analysis
Protein Concentration Method
Lowry­-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected contaminants
actin/ N-myc/ GRP78
Characterization: RNA analysis
RNA analysis
Type
(RT)­(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
5 U/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-350
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
50-350
EV210090 1/6 Simiiformes CSF (d)(U)C Yuyama, Kohei 2015 44%

Study summary

Full title
All authors
Kohei Yuyama, Hui Sun, Seigo Usuki, Shota Sakai, Hisatoshi Hanamatsu, Tetsuo Mioka, Nobuyuki Kimura, Megumi Okada, Hidetoshi Tahara, Jun-ichi Furukawa, Naoki Fujitani, Yasuro Shinohara, Yasuyuki Igarashi
Journal
FEBS Lett
Abstract
Elevated amyloid-β peptide (Aβ) in brain contributes to Alzheimer's disease (AD) pathogenesis. We (show more...)Elevated amyloid-β peptide (Aβ) in brain contributes to Alzheimer's disease (AD) pathogenesis. We demonstrated the presence of exosome-associated Aβ in the cerebrospinal fluid (CSF) of cynomolgus monkeys and APP transgenic mice. The levels of exosome-associated Aβ notably decreased in the CSF of aging animals. We also determined that neuronal exosomes, but not glial exosomes, had abundant glycosphingolipids and could capture Aβ. Infusion of neuronal exosomes into brains of APP transgenic mice decreased Aβ and amyloid depositions, similarly to what reported previously on neuroblastoma-derived exosomes. These findings highlight the role of neuronal exosomes in Aβ clearance, and suggest that their downregulation might relate to Aβ accumulation and, ultimately, the development of AD pathology. (hide)
EV-METRIC
44% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
CSF
Sample origin
Cynomolgus monkey
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Flotillin1/ GM-1/ Amyloid-β/ Alix
non-EV: Tranferrin receptor/ GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Simiiformes
Sample Type
CSF
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ GM-1/ Amyloid-β/ Alix
Not detected contaminants
Tranferrin receptor/ GM130
ELISA
Detected EV-associated proteins
Amyloid beta
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV150108 3/4 Homo sapiens MCF-7 (d)(U)C Tosar JP 2015 44%

Study summary

Full title
All authors
Tosar JP, Gámbaro F, Sanguinetti J, Bonilla B, Witwer KW, Cayota A.
Journal
Nucleic Acids Res
Abstract
Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulat (show more...)Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19-60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5' tRNA halves and 5' RNA Y4-derived fragments of 31-33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF-7
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
30
Pelleting: rotor type
Not specified
Pelleting: speed (g)
16000
Wash: time (min)
30
Wash: Rotor Type
Not specified
Wash: speed (g)
16000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Not detected EV-associated proteins
CD9/ CD63/ TSG101
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing;ddPCR
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Not Reported
NTA
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160016 1/3 Homo sapiens DKO-1 (d)(U)C
UF
Filtration
Cha DJ 2015 44%

Study summary

Full title
All authors
Cha DJ, Franklin JL, Dou Y, Liu Q, Higginbotham JN, Demory Beckler M, Weaver AM, Vickers K, Prasad N, Levy S, Zhang B, Coffey RJ, Patton JG.
Journal
Elife
Abstract
Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor mi (show more...)Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the... (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
UF
Filtration
Protein markers
EV: "Flotillin1/ TSG101/ HSP70/ KRAS/ EGFR/ RAP1/ SRC/ LYN/ ITGB1/ ITGA2/ ITGAV/ ITGA6/ ITGB4/ EPHA2/ EPS8/ CTTN"
non-EV: VDAC
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
DKO-1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Not specified
Pelleting: speed (g)
150000
Wash: time (min)
180
Wash: Rotor Type
Not specified
Wash: speed (g)
150000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
"Flotillin1/ TSG101/ HSP70/ KRAS/ EGFR/ RAP1/ SRC/ LYN/ ITGB1/ ITGA2/ ITGAV/ ITGA6/ ITGB4/ EPHA2/ EPS8/ CTTN"
Not detected contaminants
VDAC
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
"(RT)(q)PCR;RNA sequencing"
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
56.3
EV160016 3/3 Homo sapiens DKs-8 (d)(U)C
UF
Filtration
Cha DJ 2015 44%

Study summary

Full title
All authors
Cha DJ, Franklin JL, Dou Y, Liu Q, Higginbotham JN, Demory Beckler M, Weaver AM, Vickers K, Prasad N, Levy S, Zhang B, Coffey RJ, Patton JG.
Journal
Elife
Abstract
Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor mi (show more...)Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the... (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
UF
Filtration
Protein markers
EV: "Flotillin1/ TSG101/ HSP70/ EGFR/ RAP1/ SRC/ ITGB1/ ITGA2/ ITGAV/ CTNNA"
non-EV: VDAC
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
DKs-8
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Not specified
Pelleting: speed (g)
150000
Wash: time (min)
180
Wash: Rotor Type
Not specified
Wash: speed (g)
150000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
"Flotillin1/ TSG101/ HSP70/ EGFR/ RAP1/ SRC/ ITGB1/ ITGA2/ ITGAV/ CTNNA"
Not detected contaminants
VDAC
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
"(RT)(q)PCR;RNA sequencing"
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
59.2
EV150104 1/4 Homo sapiens Urine DC
(d)(U)C
Pocsfalvi G 2015 44%

Study summary

Full title
All authors
Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G.
Journal
Proteomics Clin Appl
Abstract
PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide)
EV-METRIC
44% (81st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DC
(d)(U)C
Protein markers
EV: TSG101/ Alix/ AQP2/ PKD22/ NHE3/ CD9/ PKD11
non-EV: / Uromodulin/ Albumin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
Not specified
Pelleting: speed (g)
200000
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ NHE3/ AQP2/ PKD11/ PKD22/ TSG101/ Alix
Detected contaminants
Not detected contaminants
Albumin/ Uromodulin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
95
EM
EM-type
Transmission-EM
Image type
Wide-field
EV150103 5/7 Homo sapiens HUVEC (d)(U)C Dieudé M 2015 44%

Study summary

Full title
All authors
Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ
Journal
J Transl Med
Abstract
Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Apoptosis
Focus vesicles
apoptotic body
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
127.9 (pelleting)
Protein markers
EV: Tubulin/ TCTP/ Fibronectin/ Syntenin/ LG3/ GM130
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Biomarker, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
Serum free medium
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
1080
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
127.9
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
2000
Western Blot
Detected EV-associated proteins
Syntenin, Fibronectin, TCTP, GM130, Tubulin, LG3
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BDCantoII Special Order Research Product
Hardware adjustment
This high sensitivity Flow cytometer (hsFCM) is equipped with a small particle option. The forward scatter (FSC) on this dedicated equipment is coupled to a photomultiplier tube (PMT) with a 488 nm solid state;100mW output blue laser (rather than the conventional 20 mW);and includes a 633nmHeNe;20mW output red laser and a 405 nm solid state diode;50mW output violet laser. The hsFCM includes a FSC-PMT and a Fourier optical transformation unit;which reduces the background noise and increases the angle of diffusion;therby enhancing the detection of small-diameter particles.
Calibration bead size
0.09,0.45,0.84,1,3.2
Report type
Median
Reported size (nm)
100-200
EV concentration
Yes
Particle yield
3.50E+07 particles/million cells
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV150031 1/1 Homo sapiens NAY (d)(U)C
DG
van Niel G 2015 44%

Study summary

Full title
All authors
van Niel G, Bergam P, Di Cicco A, Hurbain I, Lo Cicero A, Dingli F, Palmulli R, Fort C, Potier MC, Schurgers LJ, Loew D, Levy D, Raposo G
Journal
Cell Rep
Abstract
Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related dise (show more...)Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
Endosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Protein markers
EV: TSG101/ ApoE/ CD63
non-EV:
Proteomics
yes
EV density (g/ml)
1.06-1.15
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
10
Highest density fraction
40
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ ApoE
ELISA
Detected EV-associated proteins
ApoE
Characterization: Particle analysis
EM
EM-type
immune EM/ cryo EM
EM protein
CD63;ApoE
Image type
Close-up, Wide-field
EV150051 1/3 Homo sapiens NAY (d)(U)C Hoshino A 2015 44%

Study summary

Full title
All authors
Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, Molina H, Kohsaka S, Di Giannatale A, Ceder S, Singh S, Williams C, Soplop N, Uryu K, Pharmer L, King T, Bojmar L, Davies AE, Ararso Y, Zhang T, Zhang H, Hernandez J, Weiss JM, Dumont-Cole VD, Kramer K, Wexler LH, Narendran A, Schwartz GK, Healey JH, Sandstrom P, Jørgen Labori K, Kure EH, Grandgenett PM, Hollingsworth MA, de Sousa M, Kaur S, Jain M, Mallya K, Batra SK, Jarnagin WR, Brady MS, Fodstad O, Muller V, Pantel K, Minn AJ, Bissell MJ, Garcia BA, Kang Y, Rajasekhar VK, Ghajar CM, Matei I, Peinado H, Bromberg J, Lyden D
Journal
Nature
Abstract
Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's gr (show more...)Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins ?6?4 and ?6?1 were associated with lung metastasis, while exosomal integrin ?v?5 was linked to liver metastasis. Targeting the integrins ?6?4 and ?v?5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: GAPDH
non-EV:
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Wash: volume per pellet (ml)
20
Wash: Rotor Type
70Ti
Wash: adjusted k-factor
156.9
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
GAPDH
ELISA
Detected EV-associated proteins
GAPDH
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV150012 1/2 Homo sapiens NAY (d)(U)C Santi A 2015 44%

Study summary

Full title
All authors
Santi A, Caselli A, Ranaldi F, Paoli P, Mugnaioni C, Michelucci E, Cirri P
Journal
Biochim Biophys Acta Mol Cell Res
Abstract
Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matri (show more...)Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matrix, form the structural scaffolding of organs. In solid tumors, interaction with cancer cells induces fibroblasts transdifferentiation into an activated form, which become a fundamental part of the tumor stroma. Within tumor microenvironment stromal and cancer cells engage a crosstalk that is mediated by soluble factors, cellcell contacts and extracellular vesicles trafficlking. Here we report that fibroblasts have the ability to transfer a remarkable amount of proteins and lipids to neighboring cells, in an ectosome-dependent fashion, identifying a novel and native property of these cells. Cancer-associated fibroblasts show an enhanced production and delivering of ectc:Jsomes to cancer cells compared to normal fibroblasts. As a consequence of this phenomenon, tumor cells increase their proliferation rate, indicating that ectosome-mediated trafficking could be a relevant mechanism mediating the trophic function of activated connective tissue on tumor cells. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
Vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Tubulin/ CD81/ GAPDH
non-EV: Alpha-enolase/ Vimentin/ Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ Tubulin/ GAPDH
Detected contaminants
Cell organelle protein/ Alpha-enolase/ Vimentin
ELISA
Detected EV-associated proteins
Tubulin/ GAPDH
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Wide-field
EV150012 2/2 Homo sapiens NAY (d)(U)C Santi A 2015 44%

Study summary

Full title
All authors
Santi A, Caselli A, Ranaldi F, Paoli P, Mugnaioni C, Michelucci E, Cirri P
Journal
Biochim Biophys Acta Mol Cell Res
Abstract
Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matri (show more...)Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matrix, form the structural scaffolding of organs. In solid tumors, interaction with cancer cells induces fibroblasts transdifferentiation into an activated form, which become a fundamental part of the tumor stroma. Within tumor microenvironment stromal and cancer cells engage a crosstalk that is mediated by soluble factors, cellcell contacts and extracellular vesicles trafficlking. Here we report that fibroblasts have the ability to transfer a remarkable amount of proteins and lipids to neighboring cells, in an ectosome-dependent fashion, identifying a novel and native property of these cells. Cancer-associated fibroblasts show an enhanced production and delivering of ectc:Jsomes to cancer cells compared to normal fibroblasts. As a consequence of this phenomenon, tumor cells increase their proliferation rate, indicating that ectosome-mediated trafficking could be a relevant mechanism mediating the trophic function of activated connective tissue on tumor cells. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
Vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alpha-enolase/ Tubulin/ GAPDH/ Vimentin/ Integrin-beta1/ MMP2
non-EV: CD81/ Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
40
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Integrin-beta1/ MMP2/ Tubulin/ GAPDH/ Vimentin/ Alpha-enolase
Detected contaminants
Cell organelle protein/ CD81
ELISA
Detected EV-associated proteins
Integrin-beta1/ MMP2/ Tubulin/ GAPDH/ Vimentin/ Alpha-enolase
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up
EV150003 2/2 Homo sapiens NAY (d)(U)C Saari H 2015 44%

Study summary

Full title
All authors
Saari H, Lázaro-Ibáñez E, Viitala T, Vuorimaa-Laukkanen E, Siljander P, Yliperttula M
Journal
J Control Release
Abstract
BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate int (show more...)BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. METHODS: EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. RESULTS: Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. CONCLUSIONS: Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes / microvesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
52.47 (washing)
Protein markers
EV: TSG101/ CD63/ CD81/ GAPDH/ Alpha-tubulin/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Wash: volume per pellet (ml)
1
Wash: Rotor Type
TLA55
Wash: adjusted k-factor
52.47
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin/ GAPDH
ELISA
Detected EV-associated proteins
Alpha-tubulin/ GAPDH
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ cryo EM
Image type
Close-up
EV150015 2/2 Homo sapiens NAY (d)(U)C
DG
Filtration
Paggetti J 2015 44%

Study summary

Full title
All authors
Paggetti J, Haderk F, Seiffert M, Janji B, Distler U, Ammerlaan W, Kim YJ, Adam J, Lichter P, Solary E, Berchem G, Moussay E
Journal
Blood
Abstract
Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metast (show more...)Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected ?-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Protein markers
EV: TSG101/ Rab5a/ CD63/ HSP90/ Alix/ AChE/ HSP72/ MHC2/ Ago2
non-EV:
Proteomics
yes
EV density (g/ml)
1.15-1.17
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.2
Highest density fraction
2.5
Orientation
Top-down
Speed (g)
110000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ HSP90/ TSG101/ MHC2/ Rab5a/ HSP72/ Ago2/ AChE
ELISA
Detected EV-associated proteins
MHC2/ Rab5a/ HSP72/ Ago2/ AChE
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV150006 1/2 Homo sapiens
Mus musculus
NAY (d)(U)C
DG
Filtration
Melo SA 2015 44%

Study summary

Full title
All authors
Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R
Journal
Nature
Abstract
Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. (show more...)Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Protein markers
EV: CD81/ Flotilin1
non-EV:
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens / Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Wash: volume per pellet (ml)
35
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
150000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ Flotilin1
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ immune EM
EM protein
CD9
Image type
Close-up
EV150006 2/2 Homo sapiens Serum (d)(U)C
DG
Filtration
Melo SA 2015 44%

Study summary

Full title
All authors
Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R
Journal
Nature
Abstract
Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. (show more...)Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy. (hide)
EV-METRIC
44% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Protein markers
EV: Flotilin1
non-EV:
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
960
Wash: volume per pellet (ml)
11
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
150000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ immune EM
EM protein
CD9
Image type
Close-up
EV150007 3/10 Homo sapiens NAY (d)(U)C
Filtration
Lobb RJ 2015 44%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Adj. k-factor
157.1 (pelleting) / 157.1 (washing)
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV:
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
50.2Ti
Pelleting: adjusted k-factor
157.1
Wash: volume per pellet (ml)
1
Wash: Rotor Type
50.2Ti
Wash: adjusted k-factor
157.1
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
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