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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
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  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV150009 1/1 Homo sapiens Other 0.2 µm filter
dUC
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% (97th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Other
Focus vesicles
exosomes / microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
162 (pelleting)
Protein markers
EV: Alix/ CD63
non-EV: CD41/ CD235a/b/ CD45
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
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 Cell culture supernatant dUC
Iodixanol-DG (valid.)
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% (98th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Iodixanol-DG (valid.)
Protein markers
EV: Alix/ CD63/ TSG101
non-EV: Cell organelle protein/ Beta-actin
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
70
Density gradient
Only used for validation of main results
1
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 Cell culture supernatant 0.2 µm filter
Density cushion
dUC
Sucrose-DG (valid.)
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% (98th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Density cushion + dUC + Sucrose-DG (valid.)
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
120
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Density gradient
Only used for validation of main results
1
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
EV150034 2/2 Homo sapiens Blood plasma 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% (97th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
Vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC
Protein markers
EV: CD81/ CD9/ GAPDH
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
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
Image type
Close-up
EV150003 1/2 Homo sapiens Cell culture supernatant dUC 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% (95th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes / microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
52.47 (washing)
Protein markers
EV: CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: 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 0.2 µm filter
Density cushion
dUC
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% (97th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes / microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Density cushion + dUC
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: Alix/ CD63/ HSP70/ TSG101
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Ascites
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: 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
Particle analysis: flow cytometry
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV150002 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
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% (95th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: CD63/ CD9/ MFGE8
non-EV: None
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
90
Density gradient
Only used for validation of main results
1
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 Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
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% (95th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ TSG101/ CD82/ Rab5/ Caveolin1
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Density gradient
Only used for validation of main results
1
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 Cell culture supernatant dUC
Iodixanol-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% (95th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes / Ectosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Iodixanol-DG
Adj. k-factor
276.6 (pelleting)
Protein markers
EV: Alix/ CD63/ CD81/ TSG101
non-EV: Cell organelle protein/ MMP2
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
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 Cell culture supernatant dUC
Iodixanol-DG (valid.)
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% (95th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
microparticles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Iodixanol-DG (valid.)
Protein markers
EV: TSG101/ VDAC
non-EV: None
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
90
Density gradient
Only used for validation of main results
1
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
EV150007 1/10 Homo sapiens Cell culture supernatant Commercial
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial + UF
Protein markers
EV: Flotilin1/ HSP70/ TSG101
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Commercial kit
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 Cell culture supernatant 0.2 µm filter
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + UF
Protein markers
EV: CD63/ Flotilin1/ HSP70/ TSG101
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
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 Cell culture supernatant 0.2 µm filter
Iodixanol-DG
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Iodixanol-DG + UF
Protein markers
EV: Flotilin1/ HSP70/ TSG101
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
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 Cell culture supernatant 0.2 µm filter
Commercial
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial
Protein markers
EV: Flotilin1/ HSP70/ TSG101
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
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 Cell culture supernatant 0.2 µm filter
Commercial
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial
Protein markers
EV: Flotilin1/ HSP70/ TSG101
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
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
EV150031 1/1 Homo sapiens Cell culture supernatant dUC
Iodixanol-DG (valid.)
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
Endosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Iodixanol-DG (valid.)
Protein markers
EV: CD63/ TSG101/ ApoE
non-EV: None
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
60
Density gradient
Only used for validation of main results
1
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
Image type
Close-up, Wide-field
EV150051 1/3 Homo sapiens Cell culture supernatant dUC 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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: GAPDH
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
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 Cell culture supernatant dUC 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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
Vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD81/ Tubulin/ GAPDH
non-EV: Cell organelle protein/ Alpha-enolase/ Vimentin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: 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 Cell culture supernatant dUC 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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
Vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: Integrin-beta1/ MMP2/ Tubulin/ GAPDH/ Vimentin/ Alpha-enolase
non-EV: Cell organelle protein/ CD81
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
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 Cell culture supernatant dUC 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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes / microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
52.47 (washing)
Protein markers
EV: CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin/ GAPDH
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
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 Cell culture supernatant 0.45 µm filter
Density cushion
dUC
Sucrose-DG (valid.)
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + Density cushion + dUC + Sucrose-DG (valid.)
Protein markers
EV: Alix/ CD63/ HSP90/ TSG101/ MHC2/ Rab5a/ HSP72/ Ago2/ AChE
non-EV: None
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
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Density gradient
Only used for validation of main results
1
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
Particle analysis: flow cytometry
EV150006 1/2 Homo sapiens
Mus musculus
Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Protein markers
EV: CD81/ Flotilin1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens / Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Wash: volume per pellet (ml)
35
Density gradient
Only used for validation of main results
1
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
Image type
Close-up
EV150006 2/2 Homo sapiens Serum 0.2 µm filter
dUC
Sucrose-DG (valid.)
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Protein markers
EV: Flotilin1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
960
Wash: volume per pellet (ml)
11
Density gradient
Only used for validation of main results
1
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
Image type
Close-up
EV150007 3/10 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
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% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
157.1 (pelleting) / 157.1 (washing)
Protein markers
EV: Flotilin1/ HSP70/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
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
EV150011 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
Lazar I 2015 44%

Study summary

Full title
All authors
Lazar I, Clement E, Ducoux-Petit M, Denat L, Soldan V, Dauvillier S, Balor S, Burlet-Schiltz O, Larue L, Muller C, Nieto L
Journal
Pigm Cell Melanoma R
Abstract
Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma (show more...)Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma-specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro-migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells' aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment. (hide)
EV-METRIC
44% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: CD81/ Flotilin1/ TSG101
non-EV: None
Proteomics
yes
EV density (g/ml)
1.13-1.19
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
90
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ Flotilin1/ TSG101
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV150005 2/2 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
Nojima H 2015 44%

Study summary

Full title
All authors
Nojima H, Freeman CM, Schuster RM, Japtok L, Kleuser B, Edwards MJ, Gulbins E, Lentsch AB
Journal
J Hepatol
Abstract
BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hep (show more...)BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (hide)
EV-METRIC
44% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Protein markers
EV: CD63/ CD81/ TSG101
non-EV: Cell organelle protein/ Beta-actin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ TSG101
Detected contaminants
Cell organelle protein/ Beta-actin
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV150024 1/2 Homo sapiens Cell culture supernatant dUC Hazan-Halevy I 2015 44%

Study summary

Full title
All authors
Hazan-Halevy I, Rosenblum D, Weinstein S, Bairey O, Raanani P, Peer D
Journal
Cancer Lett
Abstract
Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatm (show more...)Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib(®)), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities is needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only a minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients' exosomes were taken up by both healthy and patients' B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by a cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies. (hide)
EV-METRIC
44% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD63/ CD81/ TSG101/ CD19
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
70
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ TSG101/ CD19
Detected contaminants
Calnexin
ELISA
Detected EV-associated proteins
CD19
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
EM
EM-type
transmission EM/ immune EM
Image type
Close-up
EV150039 1/1 Homo sapiens Other dUC
SEC
Sucrose-DG
Dubois L 2015 44%

Study summary

Full title
All authors
Dubois L, Ronquist KK, Ek B, Ronquist G, Larsson A
Journal
Mol Cell Proteomics
Abstract
Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular (show more...)Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163. (hide)
EV-METRIC
44% (85th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Other
Focus vesicles
Prostasomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + SEC + Sucrose-DG
Adj. k-factor
126 (pelleting)
Protein markers
EV: Flotilin1/ Flotillin2/ Clathrin
non-EV: None
Proteomics
yes
EV density (g/ml)
1.13-1.19
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
120
Pelleting: rotor type
90Ti
Pelleting: adjusted k-factor
126.0
Density gradient
Lowest density fraction
1
Highest density fraction
2
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ Flotillin2/ Clathrin
ELISA
Detected EV-associated proteins
Flotillin2/ Clathrin
Characterization: Particle analysis
EV150001 1/1 Homo sapiens Urine SEC
UF
Lozano-Ramos I 2015 43%

Study summary

Full title
All authors
Lozano-Ramos I, Bancu I, Oliveira-Tercero A, Armengol MP, Menezes-Neto A, Del Portillo HA, Lauzurica-Valdemoros R, Borràs FE
Journal
J Extracell Vesicles
Abstract
Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this inv (show more...)Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm-Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches. (hide)
EV-METRIC
43% (82nd 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC + UF
Protein markers
EV: CD63/ CD9
non-EV: Albumin/ Tamm-Horsfall glycoprotein
Proteomics
no
TEM measurements
80-120
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Characterization: Particle analysis
NTA
EM
EM-type
cryo EM
Image type
Close-up, Wide-field
EV150018 2/2 Mus musculus Other dUC
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Asai H 2015 43%

Study summary

Full title
All authors
Asai H, Ikezu S, Tsunoda S, Medalla M, Luebke J, Haydar T, Wolozin B, Butovsky O, Kügler S, Ikezu T
Journal
Nat Neurosci
Abstract
Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spr (show more...)Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus-based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target. (hide)
EV-METRIC
43% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Other
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Immunoaffinity (valid.) + Sucrose-DG (valid.)
Protein markers
EV: AChE
non-EV: None
Proteomics
no
TEM measurements
106.4+-29.6
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
70
Wash: volume per pellet (ml)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Immunoaffinity capture
Selected surface protein(s)
Tsg101
Western Blot
Detected EV-associated proteins
AChE
ELISA
Detected EV-associated proteins
AChE
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV150007 2/10 Homo sapiens Blood plasma Commercial
UF
Lobb RJ 2015 38%

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
38% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial + UF
Protein markers
EV: Flotilin1
non-EV: Cell organelle protein/ Albumin
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Commercial kit
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Detected contaminants
Cell organelle protein/ Albumin
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 9/10 Homo sapiens Blood plasma 0.2 µm filter
Commercial
Lobb RJ 2015 38%

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
38% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial
Protein markers
EV: Flotilin1
non-EV: Cell organelle protein/ Albumin
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Detected contaminants
Cell organelle protein/ Albumin
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV150007 10/10 Homo sapiens Blood plasma 0.2 µm filter
Commercial
Lobb RJ 2015 38%

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
38% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial
Protein markers
EV: Flotilin1
non-EV: Cell organelle protein/ Albumin
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
Exo-spin
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Detected contaminants
Cell organelle protein/ Albumin
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150005 1/2 Mus musculus Serum Commercial Nojima H 2015 38%

Study summary

Full title
All authors
Nojima H, Freeman CM, Schuster RM, Japtok L, Kleuser B, Edwards MJ, Gulbins E, Lentsch AB
Journal
J Hepatol
Abstract
BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hep (show more...)BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (hide)
EV-METRIC
38% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: CD63/ CD81/ TSG101
non-EV: Cell organelle protein/ Beta-actin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Serum
Isolation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ TSG101
Detected contaminants
Cell organelle protein/ Beta-actin
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up
EV150004 2/2 Homo sapiens Cell culture supernatant 0.2 µm filter
Commercial
UF
Clark DJ 2015 38%

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
38% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial + UF
Protein markers
EV: Alix/ CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63
Characterization: Particle analysis
EV150013 1/1 Mus musculus Cell culture supernatant 0.2 µm filter
Commercial
dUC
Zhu Y 2015 33%

Study summary

Full title
All authors
Zhu Y, Chen X, Pan Q, Wang Y, Su S, Jiang C, Li Y, Xu N, Wu L, Lou X, Liu S
Journal
J Proteome Res
Abstract
Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extrac (show more...)Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment. (hide)
EV-METRIC
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial + dUC
Protein markers
EV: Alix/ HSP90/ TSG101
non-EV: Cell organelle protein
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ HSP90/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV150033 1/1 Mus musculus Cell culture supernatant dUC DeClercq V 2015 33%

Study summary

Full title
All authors
DeClercq V, D'Eon B, McLeod RS
Journal
Biochim Biophys Act Cell Biol L
Abstract
Little is known about the effects of fatty acids on adiponectin oligomer assembly and trafficking. T (show more...)Little is known about the effects of fatty acids on adiponectin oligomer assembly and trafficking. The aim of this study was to examine the effects of different fatty acids on adiponectin transport and secretion in differentiated 3T3-L1 adipocytes. Subcellular fractionation and immunofluorescence microscopy revealed that the majority of cellular adiponectin was located in the endoplasmic reticulum (ER). Adiponectin secretion was increased by treatment with fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and several fatty acids changed the cellular localization of adiponectin. Adiponectin secretion has been shown to be altered by ER stress and interactions with ER chaperone proteins. However these mechanisms were not influenced by fatty acids, suggesting that alternative mechanisms must be responsible for the increased secretion of adiponectin observed with fatty acid treatment. Secretion of adiponectin was blocked by Brefeldin A, but we identified a minor pool of adiponectin that could be secreted from beyond the Brefeldin A block. Exosomes appeared to contribute to a minor amount of adiponectin secreted from the cell, and exosome release was increased by treatment with DHA. These data suggest that the ER is an important site of adiponectin accumulation and that treatment with long chain omega-3 fatty acids increases adiponectin release. Furthermore, the secretory pathway of adiponectin is complex, involving both the classical ER-Golgi pathway as well as unconventional secretory mechanisms such as an exosome-mediated pathway. (hide)
EV-METRIC
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: Alix/ TSG101
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
90
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EV150032 1/1 Bos bovis Milk 0.2 µm filter
dUC
Wolf T 2015 33%

Study summary

Full title
All authors
Wolf T, Baier SR, Zempleni J
Journal
J Nutr
Abstract
BACKGROUND: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs i (show more...)BACKGROUND: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs in tissues and body fluids is encapsulated in exosomes, thereby conferring protection against degradation and a pathway for intestinal transport. MicroRNAs in cow milk are bioavailable in humans. OBJECTIVE: This research assessed the transport mechanism of bovine milk exosomes, and therefore microRNAs, in human and rodent intestinal cells. METHODS: The intestinal transport of bovine milk exosomes and microRNAs was assessed using fluorophore-labeled bovine milk exosomes in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. Transport kinetics and mechanisms were characterized using dose-response studies, inhibitors of vesicle transport, carbohydrate competitors, proteolysis of surface proteins on cells and exosomes, and transepithelial transport in transwell plates. RESULTS: Exosome transport exhibited saturation kinetics at 37°C [Michaelis constant (Km) = 55.5 ± 48.6 ?g exosomal protein/200 ?L of media; maximal transport rate = 0.083 ± 0.057 ng of exosomal protein · 81,750 cells(-1) · h(-1)] and decreased by 64% when transport was measured at 4°C, consistent with carrier-mediated transport in Caco-2 cells. Exosome uptake decreased by 61-85% under the following conditions compared with controls in Caco-2 cells: removal of exosome and cell surface proteins by proteinase K, inhibition of endocytosis and vesicle trafficking by synthetic inhibitors, and inhibition of glycoprotein binding by carbohydrate competitors. When milk exosomes, at a concentration of 5 times the Km, were added to the upper chamber in transwell plates, Caco-2 cells accumulated miR-29b and miR-200c in the lower chamber, and reverse transport was minor. Transport characteristics were similar in IEC-6 cells and Caco-2 cells, except that substrate affinity and transporter capacity were lower and higher, respectively. CONCLUSION: The uptake of bovine milk exosomes is mediated by endocytosis and depends on cell and exosome surface glycoproteins in human and rat intestinal cells. (hide)
EV-METRIC
33% (65th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Milk
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
176.2 (pelleting)
Protein markers
EV: CD63
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Bos bovis
Sample Type
Milk
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
F37L-8x100
Pelleting: adjusted k-factor
176.2
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV150054 1/1 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
Wang X 2015 33%

Study summary

Full title
All authors
Wang X, Gu H, Qin D, Yang L, Huang W, Essandoh K, Wang Y, Caldwell CC, Peng T, Zingarelli B, Fan GC
Journal
Sci Rep
Abstract
Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However (show more...)Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis. (hide)
EV-METRIC
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
206.6 (pelleting)
Protein markers
EV: CD63/ CD81
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
70
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
206.6
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81
Characterization: Particle analysis
DLS
EM
EV150030 1/1 Homo sapiens Urine dUC Perez-Hernandez J 2015 33%

Study summary

Full title
All authors
Perez-Hernandez J, Forner MJ, Pinto C, Chaves FJ, Cortes R, Redon J
Journal
PLoS One
Abstract
There is increased interest in using microRNAs (miRNAs) as biomarkers in different diseases. Present (show more...)There is increased interest in using microRNAs (miRNAs) as biomarkers in different diseases. Present in body fluids, it is controversial whether or not they are mainly enclosed in exosomes, thus we studied if urinary miRNAs are concentrated inside exosomes and if the presence of systemic lupus erythematosus with or without lupus nephritis modifies their distribution pattern. We quantified specific miRNAs in urine of patients with systemic lupus erythematosus (n = 38) and healthy controls (n = 12) by quantitative reverse-transcription PCR in cell-free urine, exosome-depleted supernatant and exosome pellet obtained by ultracentrifugation. In control group, miR-335 (hide)
EV-METRIC
33% (71st 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
27.21 (pelleting)
Protein markers
EV: CD9/ TSG101
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
70
Pelleting: rotor type
70.1Ti
Pelleting: adjusted k-factor
27.21
Wash: volume per pellet (ml)
2
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV150029 1/1 Homo sapiens Urine 0.2 µm filter
dUC
UF
Øverbye A 2015 33%

Study summary

Full title
All authors
Øverbye A, Skotland T, Koehler CJ, Thiede B, Seierstad T, Berge V, Sandvig K, Llorente A
Journal
Oncotarget
Abstract
Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-spe (show more...)Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-specific molecules can be found in exosomes isolated from biological fluids. We have here investigated the proteome of urinary exosomes by using mass spectrometry to identify proteins differentially expressed in prostate cancer patients compared to healthy male controls. In total, 15 control and 16 prostate cancer samples of urinary exosomes were analyzed. Importantly, 246 proteins were differentially expressed in the two groups. The majority of these proteins (221) were up-regulated in exosomes from prostate cancer patients. These proteins were analyzed according to specific criteria to create a focus list that contained 37 proteins. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. Even though several of these proteins showed high sensitivity and specificity for prostate cancer as individual biomarkers, combining them in a multi-panel test has the potential for full differentiation of prostate cancer from non-disease controls. The highest sensitivity, 94%, was observed for transmembrane protein 256 (TM256; chromosome 17 open reading frame 61). LAMTOR proteins were also distinctly enriched with very high specificity for patient samples. TM256 and LAMTOR1 could be used to augment the sensitivity to 100%. Other prominent proteins were V-type proton ATPase 16 kDa proteolipid subunit (VATL), adipogenesis regulatory factor (ADIRF), and several Rab-class members and proteasomal proteins. In conclusion, this study clearly shows the potential of using urinary exosomes in the diagnosis and clinical management of prostate cancer. (hide)
EV-METRIC
33% (71st 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + UF
Adj. k-factor
156.9 (pelleting) / 276.6 (washing)
Protein markers
EV: CD81/ CD9/ TSG101
non-EV: Tamm-Horsfall glycoprotein
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
70
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Wash: Rotor Type
SW40
Wash: adjusted k-factor
276.6
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ CD9/ TSG101
Detected contaminants
Tamm-Horsfall glycoprotein
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Wide-field
EV150007 6/10 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Iodixanol-DG
Lobb RJ 2015 33%

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
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Iodixanol-DG
Adj. k-factor
157.1 (pelleting) / 157.1 (washing)
Protein markers
EV: Flotilin1/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
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
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/ TSG101
Characterization: Particle analysis
TRPS
EV150028 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Koch R 2015 33%

Study summary

Full title
All authors
Koch R, Aung T, Vogel D, Chapuy B, Wenzel D, Becker S, Sinzig U, Venkataramani V, von Mach T, Jacob R, Truemper L, Wulf GG
Journal
Clin Cancer Res
Abstract
PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with agg (show more...)PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with aggressive B-cell lymphoma, tumor cell susceptibility to chemotherapy varies, with mostly fatal outcome in cases of resistant disease. We and others have shown before that export of cytostatic drugs contributes to drug resistance. Now we provide a novel approach to overcome exosome-mediated drug resistance in aggressive B-cell lymphomas. EXPERIMENTAL DESIGN: We used well-established centrifugation protocols to purify exosomes from DLBCL cell lines and detected anthracyclines using FACS and HPLC. We used shRNA knockdown of ABCA3 to determine ABCA3 dependence of chemotherapy susceptibility and monitored ABCA3 expression after indomethacin treatment using qPCR. Finally, we established an in vivo assay using a chorioallantoic membrane (CAM) assay to determine the synergy of anthracycline and indomethacin treatment. RESULTS: We show increased efficacy of the anthracycline doxorubicin and the anthracenedione pixantrone by suppression of exosomal drug resistance with indomethacin. B-cell lymphoma cells in vitro efficiently extruded doxorubicin and pixantrone, in part compacted in exosomes. Exosomal biogenesis was critically dependent on the expression of the ATP-transporter A3 (ABCA3). Genetic or chemical depletion of ABCA3 augmented intracellular retention of both drugs and shifted the subcellular drug accumulation to prolonged nuclear retention. Indomethacin increased the cytostatic efficacy of both drugs against DLBCL cell lines in vitro and in vivo in a CAM assay. CONCLUSIONS: We propose pretreatment with indomethacin toward enhanced antitumor efficacy of anthracyclines and anthracenediones. Clin Cancer Res; 1-10. ©2015 AACR. (hide)
EV-METRIC
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
130.7 (pelleting)
Protein markers
EV: CD63/ CD81/ CD9/ "ADAM10/ Flotillin2/ GAPDH"
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
130.7
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ "ADAM10/ Flotillin2/ GAPDH"
ELISA
Detected EV-associated proteins
"ADAM10/ Flotillin2/ GAPDH"
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV150026 1/1 Caenorhabditis elegans Cell culture supernatant dUC
UF
Hyenne V 2015 33%

Study summary

Full title
All authors
Hyenne V, Apaydin A, Rodriguez D, Spiegelhalter C, Hoff-Yoessle S, Diem M, Tak S, Lefebvre O, Schwab Y, Goetz JG, Labouesse M
Journal
J Cell Biol
Abstract
Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plas (show more...)Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1-deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release. (hide)
EV-METRIC
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + UF
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: Alix/ CD63/ TSG101/ HSC70
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Caenorhabditis elegans
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
70
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ TSG101/ HSC70
ELISA
Detected EV-associated proteins
HSC70
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV150041 1/1 Homo sapiens Cell culture supernatant dUC Lamichhane TN 2015 33%

Study summary

Full title
All authors
Lamichhane TN, Raiker RS, Jay SM
Journal
Mol Pharm
Abstract
Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delive (show more...)Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles. (hide)
EV-METRIC
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: Alix
non-EV: GAPDH
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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: time(min)
120
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Wash: volume per pellet (ml)
29
Wash: Rotor Type
70Ti
Wash: adjusted k-factor
156.9
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix
Detected contaminants
GAPDH
Characterization: Particle analysis
NTA
EV150014 1/2 Homo sapiens Cell culture supernatant 0.2 µm filter
SEC
UF
Grasso L 2015 33%

Study summary

Full title
All authors
Grasso L, Wyss R, Weidenauer L, Thampi A, Demurtas D, Prudent M, Lion N, Vogel H
Journal
Anal Bioanal Chem
Abstract
We report on a generic method to detect and identify the molecular profile of exosomes either derive (show more...)We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use. (hide)
EV-METRIC
33% (75th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + SEC + UF
Protein markers
EV: CD63/ CD9/ CD44/ CD24/ EpCAM
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD44/ CD24/ EpCAM
ELISA
Detected EV-associated proteins
CD63/ CD9/ CD44/ CD24/ EpCAM
Characterization: Particle analysis
DLS
EM
EM-type
cryo EM
Image type
Close-up
EV150014 2/2 Homo sapiens Blood plasma 0.2 µm filter
SEC
Grasso L 2015 33%

Study summary

Full title
All authors
Grasso L, Wyss R, Weidenauer L, Thampi A, Demurtas D, Prudent M, Lion N, Vogel H
Journal
Anal Bioanal Chem
Abstract
We report on a generic method to detect and identify the molecular profile of exosomes either derive (show more...)We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases suc