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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.
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
Experiment number
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Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV160001 1/1 Mus musculus Cell culture supernatant DG
dUC
Stremersch S 2016 87%

Study summary

Full title
All authors
Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC, Raemdonck K
Journal
J Control Release
Abstract
Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natu (show more...)Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier. (hide)
EV-METRIC
87% (99th 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
exosome-like 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
DG + dUC
Protein markers
EV: CD63/ CD81/ HSP70/ beta-actin
non-EV: Calreticulin/ GM130
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
B16F10, JAWSII
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Ultrafiltration (MWCO 300 kDa)
Cell viability
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
0.125
Highest density fraction
0.5
Sample volume (mL)
1
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 55 Ti
Speed (g)
200000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
150
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
115.5
Pelleting-wash: volume per pellet (mL)
5
Pelleting-wash: duration (min)
70
Pelleting-wash: rotor type
115.5
Pelleting-wash: speed (g)
SW 55 Ti
Pelleting-wash: adjusted k-factor
115.5
EV-subtype
Used subtypes
NO
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, HSP70, beta-actin
Flow cytometry specific beads
Selected surface protein(s)
CD63
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-300
EV concentration
Yes
Particle yield
1000
EM
EM-type
Cryo-EM
Image type
Close-up
EV160004 2/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 66%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
66% (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
Synovial fluid
Focus vesicles
extracellular vesicle
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
DG + dUC
Adj. k-factor
71.08 (pelleting)
Protein markers
EV: Annexin-A1/ CD90/Thy1.1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
71.08
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0M
Highest density fraction
1.4M
Sample volume (mL)
1.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
60
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
138.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Annexin-A1, CD90/Thy1.1
Fluorescent NTA
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.
EV160004 5/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 66%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
66% (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
Synovial fluid
Focus vesicles
extracellular vesicle
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
DG + dUC
Adj. k-factor
1421 (pelleting)
Protein markers
EV: Annexin-A1/ CD90/Thy1.1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
10000
Pelleting: adjusted k-factor
1421.
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0M
Highest density fraction
1.4M
Sample volume (mL)
1.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
60
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
138.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Annexin-A1, CD90/Thy1.1
Fluorescent NTA
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.
EV160008 1/3 Rattus norvegicus Cell culture supernatant dUC Cianciaruso C 2016 55%

Study summary

Full title
All authors
Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S
Journal
Diabetes
Abstract
The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide)
EV-METRIC
55% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 vesicle
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: CD9/ Flotillin-1/ GAD65/ GAD67/ IA-2/ PDI/ Insulin
non-EV: Calreticulin/ Gp96
Proteomics
yes
Show all info
Study aim
Function, Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Primary pancreatic islets
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
87.5
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: speed (g)
110000
Wash: time (min)
70
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1, GAD65, GAD67, IA-2, PDI
Not detected contaminants
Calreticulin, Gp96
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mode
Reported size (nm)
139
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
120
EV160005 1/2 Homo sapiens Bronchoalveolar lavage fluid dUC
Filtration
Héliot A 2016 55%

Study summary

Full title
All authors
Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ
Journal
Int J Hyg Environ Health
Abstract
Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide)
EV-METRIC
55% (33rd 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
Bronchoalveolar lavage fluid
Focus vesicles
extracellular vesicle
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 + Filtration
Adj. k-factor
126 (pelleting) / 126 (washing)
Protein markers
EV: CD9/ CD63/ CD81
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Bronchoalveolar lavage fluid
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
126.0
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
110000
Wash: adjusted k-factor
126.0
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, CD81
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution;Mean
Reported size (nm)
138.1±2.2
EV concentration
Yes
Particle yield
2.48E+11 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-200
Extra information
UC rotors added to the EV-TRACK entry after publication
EV160005 2/2 Homo sapiens Bronchoalveolar lavage fluid dUC
Filtration
Héliot A 2016 55%

Study summary

Full title
All authors
Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ
Journal
Int J Hyg Environ Health
Abstract
Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide)
EV-METRIC
55% (33rd 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
Bronchoalveolar lavage fluid
Focus vesicles
extracellular vesicle
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 + Filtration
Adj. k-factor
126 (pelleting) / 126 (washing)
Protein markers
EV: CD9/ CD63/ CD81
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Bronchoalveolar lavage fluid
Sample Condition
Smokers
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
126.0
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
110000
Wash: adjusted k-factor
126.0
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, CD81
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution;Mean
Reported size (nm)
139.8±3.3
EV concentration
Yes
Particle yield
1.94E+11 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-200
Extra information
UC rotors added to the EV-TRACK entry after publication
EV160004 1/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (50th 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
Synovial fluid
Focus vesicles
extracellular vesicle
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
DG + dUC
Adj. k-factor
83.68 (pelleting)
Protein markers
EV: CD9/ CD44
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
83.68
Density gradient
Density medium
Iodixanol
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Fluorescent NTA
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV160004 3/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (50th 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
Synovial fluid
Focus vesicles
extracellular vesicle
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
DG + dUC
Adj. k-factor
167.3 (pelleting)
Protein markers
EV: CD9/ CD44
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
167.3
Density gradient
Density medium
Iodixanol
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Fluorescent NTA
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV160004 4/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (50th 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
Synovial fluid
Focus vesicles
extracellular vesicle
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
DG + dUC
Adj. k-factor
1673 (pelleting)
Protein markers
EV: CD9/ CD44
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
35
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
10000
Pelleting: adjusted k-factor
1673.
Density gradient
Density medium
Iodixanol
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Fluorescent NTA
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV160007 1/3 Homo sapiens Blood plasma dUC
Filtration
SEC
Hong CS 2016 50%

Study summary

Full title
All authors
Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL
Journal
J Extracell Vesicles
Abstract
OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (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
Blood plasma
Focus vesicles
exosome
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 + Filtration + SEC
Protein markers
EV: CD9/ TSG101/ CD44/ CD34/ CD123/ CD96/ CLL-1/ Pro-TGFbeta1LAP/ PD-1/ PD-L1
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Acute myeloid leukemia
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
66
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, TSG101, CD44, CD34, CD123, CD96, CLL-1, Pro-TGFbeta1 LAP, PD-1, PD-L1
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
77-92
EV concentration
Yes
Particle yield
8.90E+10 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160007 2/3 Homo sapiens Blood plasma dUC
Filtration
SEC
Hong CS 2016 50%

Study summary

Full title
All authors
Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL
Journal
J Extracell Vesicles
Abstract
OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (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
Blood plasma
Focus vesicles
exosome
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 + Filtration + SEC
Protein markers
EV: CD9/ TSG101/ HSP70/ Cox2/ CD73/ CD39/ Fas/ FasL/ PD-1/ PD-L1
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Head and neck squamous cell carcinoma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
123
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, TSG101, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-1, PD-L1
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
73-76
EV concentration
Yes
Particle yield
1.10E+11 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160007 3/3 Homo sapiens Blood plasma dUC
Filtration
SEC
Hong CS 2016 50%

Study summary

Full title
All authors
Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL
Journal
J Extracell Vesicles
Abstract
OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (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
Blood plasma
Focus vesicles
exosome
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 + Filtration + SEC
Protein markers
EV: CD9/ TSG101/ CD123/ CLL-1/ Pro-TGFbeta1LAP/ PD-1/ HSP70/ Cox2/ CD73/ CD39/ Fas/ FasL/ PD-L1
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
32
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, TSG101, CD123, CLL-1, Pro-TGFbeta1 LAP, PD-1, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-L1
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
69-76
EV concentration
Yes
Particle yield
7.00E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160000 1/1 Homo sapiens Milk DG
dUC
van Herwijnen MJ 2016 50%

Study summary

Full title
All authors
van Herwijnen MJ, Zonneveld MI, Goerdayal S, Nolte-'t Hoen EN, Garssen J, Stahl B, Maarten Altelaar AF, Redegeld FA, Wauben MH
Journal
Mol Cell Proteomics
Abstract
Breast milk contains several macromolecular components with distinctive functions, whereby milk fat (show more...)Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. (hide)
EV-METRIC
50% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
extracellular vesicle
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
DG + dUC
Protein markers
EV: CD9/ Flotillin-1
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Milk
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Sample volume (mL)
6.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900-1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
38.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
PMID previous EV protein analysis
25206958
Extra characterization
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1
Proteomics database
Yes
Fluorescent NTA
PMID previous EV particle analysis
25206958
Extra information
A different gradient protocol was used in the publication of the same group that was referred to for additional protein and particle analysis of milk-derived extracellular vesicles (PMID: 25206958).
EV160009 1/2 Homo sapiens Cell culture supernatant dUC
dUC
Filtration
Benedikter BJ 2016 44%

Study summary

Full title
All authors
Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM
Journal
J Cell Sci
Abstract
INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 vesicle / exosome
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 + dUC + Filtration
Adj. k-factor
133.2 (pelleting)
Protein markers
EV: CD63
non-EV: grp94
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Exposed to cigarette smoke extract
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
75
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected contaminants
grp94
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
60-300
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
50-160
EV160009 2/2 Homo sapiens Cell culture supernatant dUC
dUC
Filtration
Benedikter BJ 2016 44%

Study summary

Full title
All authors
Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM
Journal
J Cell Sci
Abstract
INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 vesicle / exosome
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 + dUC + Filtration
Adj. k-factor
133.2 (pelleting)
Protein markers
EV: CD63
non-EV: grp94
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
100
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected contaminants
grp94
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
60-300
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
50-160
EV160008 2/3 Homo sapiens Cell culture supernatant dUC Cianciaruso C 2016 44%

Study summary

Full title
All authors
Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S
Journal
Diabetes
Abstract
The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 vesicle
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: CD9/ Flotillin-1/ GAD65/ IA-2/ Insulin
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Primary human pancreatic islets, primary rat pancreatic islets, INS1 cell line
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
87.5
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: speed (g)
110000
Wash: time (min)
70
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1, GAD65, IA-2
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mode
Reported size (nm)
143
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
120
EV160002 2/3 Homo sapiens Extruded cells (NIH3T3) DG
Sequential extrusion
Lunavat TR 2016 37%

Study summary

Full title
All authors
Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J
Journal
Biomaterials
Abstract
To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide)
EV-METRIC
37% (50th 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
Extruded cells (NIH3T3)
Focus vesicles
Nanovesicles
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
DG + Sequential extrusion
Protein markers
EV: PDGFR/ Flotillin-1/ CD9
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function, New methodological development
Sample
Species
Homo sapiens
Sample Type
Extruded cells (NIH3T3)
Sample Condition
Control condition
Isolation Method
Density cushion
Density medium
Iodixanol
Other
Name other isolation method
Sequential extrusion
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
PDGFR, Flotillin-1, CD9
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
180-200
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160002 3/3 Homo sapiens Extruded cells (NIH3T3) DG
Sequential extrusion
Lunavat TR 2016 37%

Study summary

Full title
All authors
Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J
Journal
Biomaterials
Abstract
To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide)
EV-METRIC
37% (50th 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
Extruded cells (NIH3T3)
Focus vesicles
Nanovesicles
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
DG + Sequential extrusion
Protein markers
EV: PDGFR/ Flotillin-1/ CD9
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function, New methodological development
Sample
Species
Homo sapiens
Sample Type
Extruded cells (NIH3T3)
Sample Condition
cMyc shRNA
Isolation Method
Density cushion
Density medium
Iodixanol
Other
Name other isolation method
Sequential extrusion
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
PDGFR, Flotillin-1, CD9
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
180-200
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160008 3/3 Rattus norvegicus Cell culture supernatant dUC Cianciaruso C 2016 33%

Study summary

Full title
All authors
Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S
Journal
Diabetes
Abstract
The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (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
Cell culture supernatant
Focus vesicles
extracellular vesicle
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
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
INS1
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
87.5
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: speed (g)
110000
Wash: time (min)
70
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix
Proteomics database
No
Fluorescent NTA
EV160006 1/1 Homo sapiens Cell culture supernatant dUC
Filtration
Holder B 2016 22%

Study summary

Full title
All authors
Holder B, Jones T, Sancho Shimizu V, Rice TF, Donaldson B, Bouqueau M, Forbes K, Kampmann B
Journal
Traffic
Abstract
During pregnancy, the placenta forms the interface between mother and fetus. Highly controlled regul (show more...)During pregnancy, the placenta forms the interface between mother and fetus. Highly controlled regulation of trans-placental trafficking is therefore essential for the healthy development of the growing fetus. Extracellular vesicle-mediated transfer of protein and nucleic acids from the human placenta into the maternal circulation is well documented; the possibility that this trafficking is bi-directional has not yet been explored but could affect placental function and impact on the fetus.We hypothesized that the ability of the placenta to respond to maternal inflammatory signals is mediated by the interaction of maternal immune cell exosomes with placental trophoblast. Utilizing the BeWo cell line and whole placental explants, we demonstrated that the human placenta internalizes macrophage-derived exosomes in a time- and dose-dependent manner. This uptake was via clathrin-dependent endocytosis. Furthermore, macrophage exosomes induced release of proinflammatory cytokines by the placenta. Taken together, our data demonstrates that exosomes are actively transported into the human placenta and that exosomes from activated immune cells modulate placental cytokine production. This represents a novel mechanism by which immune cells can signal to the placental unit, potentially facilitating responses to maternal inflammation and infection, and thereby preventing harm to the fetus. (hide)
EV-METRIC
22% (53rd 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
exosome
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 + Filtration
Protein markers
EV: Alix/ CD81
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
PMA-stimulated
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Commercial EDS
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD81
Not detected contaminants
Calnexin
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
137.6+-18.5
EV concentration
Yes
Extra information
I think that recording how EVs were labelled (including controls), and how EVs were stored prior to functional analysis (e.g. fresh/frozen) would be a good addition to this
EV160003 1/1 Mus musculus Cell culture supernatant dUC Morales-Kastresana A 2016 14%

Study summary

Full title
All authors
Morales-Kastresana A, Telford B, Musich TA, McKinnon K, Clayborne C, Braig Z, Rosner A, Demberg T, Watson DC, Karpova TS, Freeman GJ, DeKruyff RH, Pavlakis GN, Terabe M, Robert-Guroff M, Berzofsky JA, Jones JC
Journal
Sci Rep
Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that ar (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease. (hide)
EV-METRIC
14% (42nd 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 vesicle
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) / 41.45 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
DC2.4
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
70
Wash: Rotor Type
TLA-120.1
Wash: speed (g)
100000
Wash: adjusted k-factor
41.45
Protein Concentration Method
BCA
Protein Concentration
1.186
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
80-180
EV concentration
Yes
Particle yield
4.8E+11 particles/ml start sample
Particle analysis: flow cytometry
Flow cytometer type
Beckman Astrios EQ
Hardware adjustment
NanoFACS system
Calibration bead size
0.1;0.2;0.5
EV160002 1/3 Homo sapiens Extruded cells (U973) DG
Sequential extrusion
Lunavat TR 2016 0%

Study summary

Full title
All authors
Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J
Journal
Biomaterials
Abstract
To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide)
EV-METRIC
0% (median: 0% 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
Extruded cells (U973)
Focus vesicles
Nanovesicles
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
DG + Sequential extrusion
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function, New methodological development
Sample
Species
Homo sapiens
Sample Type
Extruded cells (U973)
Sample Condition
Control condition
Isolation Method
Density cushion
Density medium
Iodixanol
Other
Name other isolation method
Sequential extrusion
Protein Concentration Method
Bradford
Fluorescent NTA
Characterization: Particle analysis
DLS
Report type
Modus
Reported size (nm)
150
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