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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190055 4/4 Homo sapiens Cell culture supernatant DG
dUC
Ferreira JV 2019 67%

Study summary

Full title
All authors
Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P.
Journal
PLoS One
Abstract
Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Microvesicles (MVs)
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Protein markers
EV: CD63/ GAPDH/ CANX/ Na/K A1 ATPase
non-EV:
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
ARPE-19
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
38
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
16
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
8
Sample volume (mL)
0.5
Orientation
Bottom-up
Rotor type
Type 70.1Ti
Speed (g)
210000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
38
Pelleting: duration (min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
>200
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
GAPDH/ Na/K A1 ATPase/ CANX/ CD63
NA
EM
EM-type
Transmission-EM
Image type
Close-up
EV190040 7/12 Homo sapiens Cell culture supernatant DG
UF
Geeurickx E 2019 67%

Study summary

Full title
All authors
Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A.
Journal
Nat Commun
Abstract
Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + UF
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.076 1.088 g/ml
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MCF7
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
11
Lowest density fraction
6
Highest density fraction
18
Total gradient volume, incl. sample (mL)
15
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
186700
Duration (min)
116
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
EV-subtype
Distinction between multiple subtypes
Density
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Characterization: Particle analysis
NA
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190038 1/3 Homo sapiens Blood plasma dUC García-Silva S 2019 67%

Study summary

Full title
All authors
García-Silva S, Benito-Martín A, Sánchez-Redondo S, Hernández-Barranco A, Ximénez-Embún P, Nogués L, Mazariegos MS, Brinkmann K, Amor López A, Meyer L, Rodríguez C, García-Martín C, Boskovic J, Letón R, Montero C, Robledo M, Santambrogio L, Sue Brady M, Szumera-Ciećkiewicz A, Kalinowska I, Skog J, Noerholm M, Muñoz J, Ortiz-Romero PL, Ruano Y, Rodríguez-Peralto JL, Rutkowski P, Peinado H.
Journal
J Exp Med
Abstract
Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The asse (show more...)Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse. (hide)
EV-METRIC
67% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Melanoma patients stage III
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD63/ GADPH/ CD81/ HSP90/ TRP2/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Melanoma patients stage III
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 50.4 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
3
Wash: time (min)
70
Wash: Rotor Type
Type 50.4 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ GADPH/ CD81/ TRP2
Not detected EV-associated proteins
HSP90
Proteomics database
ProteomeXchange Consortium
Characterization: Particle analysis
NA
NTA
Report type
Median
Reported size (nm)
126
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30.4
EV190033 1/2 Homo sapiens Blood plasma dUC
Filtration
Li Min 2019 67%

Study summary

Full title
All authors
Li Min, Shengtao Zhu, Lei Chen, Xiang Liu, Rui Wei, Libo Zhao, Yuqing Yang, Zheng Zhang, Guanyi Kong, Peng Li & Shutian Zhang
Journal
J Extracell Vesicles
Abstract
Early diagnosis of colon cancer (CC) is clinically important, as it can significantly improve patien (show more...)Early diagnosis of colon cancer (CC) is clinically important, as it can significantly improve patients’ survival rate and quality of life. Although the potential role for small extracellular vesicles (sEVs) in early detection of many diseases has been repeatedly mentioned, systematic screening of plasma sEVs derived early CC specific biomarkers has not yet been reported. In this work, plasma sEVs enriched fractions were derived from 15 early-stage (TisN0M0) CC patients and 10 normal controls (NC). RNA sequencing identified a total number of 95 sEVs enriched fraction derived miRNAs with differential expression between CC and NC, most of which (60/95) was in well accordance with tissue results in the Cancer Genome Atlas (TCGA) dataset. Among those miRNAs, we selected let-7b-3p, miR-139-3p, miR-145-3p, and miR-150-3p for further validation in an independent cohort consisting of 134 participants (58 CC and 76 NC). In the validation cohort, the AUC of 4 individual miRNAs ranged from 0.680 to 0.792. A logistic model combining two miRNAs (i.e. let-7b-3p and miR-145-3p) achieved an AUC of 0.901. Adding the 3rd miRNA into this model can further increase the AUC to 0.927. Side by side comparison revealed that sEVs miRNA profile outperformed cell-free plasma miRNA in the diagnosis of early CC. In conclusion, we suggested that circulating sEVs enriched fractions have a distinct miRNA profile in CC patients, and sEVs derived miRNA could be used as a promising biomarker to detect CC at an early stage. (hide)
EV-METRIC
67% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Protein markers
EV: TSG101/ Alix/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
240
Pelleting: rotor type
P50AT2-986
Pelleting: speed (g)
150000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
P50A3-0099
Wash: speed (g)
150000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ Alix
Not detected contaminants
Calnexin
Characterization: RNA analysis
Proteinase treatment
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
100
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.01
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
75-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
EV180050 1/6 Homo sapiens Cell culture supernatant dUC
Filtration
Alice Gualerzi 2019 66%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
liver stem cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: 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
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
Other
PMID previous EV particle analysis
Other
NTA
Report type
Mean
Reported size (nm)
189 ± 27
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV180050 2/6 Homo sapiens Cell culture supernatant dUC
Filtration
Alice Gualerzi 2019 66%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: CD81/ Flotillin-1/ CD63
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
bone marrow-derived mesenchymal stem cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, Flotillin-1
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
Other
PMID previous EV particle analysis
Other
NTA
Report type
Mean
Reported size (nm)
212 ± 34
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV180050 5/6 Homo sapiens Cell culture supernatant dUC
Filtration
Alice Gualerzi 2019 66%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: CD81/ Flotillin-1/ CD63
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
bone marrow-derived mesenchymal stem cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: 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
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, Flotillin-1
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
Other
PMID previous EV particle analysis
Other
NTA
Report type
Mean
Reported size (nm)
204 ± 42
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV180050 6/6 Homo sapiens Cell culture supernatant dUC
Filtration
Alice Gualerzi 2019 66%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
liver stem cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
Other
PMID previous EV particle analysis
Other
NTA
Report type
Mean
Reported size (nm)
184 ± 33
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV180029 1/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
144.8
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 2/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
88
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV180029 3/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
178.3
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 4/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
111
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 5/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
122.7
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV180029 6/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
118.7
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 7/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
148.8
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 8/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
Electron microscopy
PMID previous EV particle analysis
Electron microscopy
NTA
Report type
Mean
Reported size (nm)
121.4
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180021 1/4 Homo sapiens Cell culture supernatant dUC Bachurski, Daniel 2019 66%

Study summary

Full title
All authors
Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann
Journal
J Extracell Vesicles
Abstract
The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide)
EV-METRIC
66% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
892 (washing)
Protein markers
EV: TSG101/ HSP70/ CD63/ CD9/ CD81
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
L540
EV-harvesting Medium
Serum free medium
Cell viability
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Pelleting: speed (g)
10000
Wash: time (min)
30
Wash: Rotor Type
TLA-55
Wash: speed (g)
10000
Wash: adjusted k-factor
892.0
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101
Not detected contaminants
Calnexin
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
100-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-200
Other particle analysis name(1)
ExoView
Report type
Size range/distribution
Report size
50-200
EV-concentration
No
Extra information
EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView
EV180021 2/4 Homo sapiens Serum dUC Bachurski, Daniel 2019 66%

Study summary

Full title
All authors
Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann
Journal
J Extracell Vesicles
Abstract
The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide)
EV-METRIC
66% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
892 (washing)
Protein markers
EV: HSP70/ CD63/ CD9
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Pelleting: speed (g)
10000
Wash: time (min)
30
Wash: Rotor Type
TLA-55
Wash: speed (g)
10000
Wash: adjusted k-factor
892.0
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70
Not detected contaminants
Calnexin
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
100-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-500
Other particle analysis name(1)
ExoView
Report type
Size range/distribution
Report size
50-200
EV-concentration
No
Extra information
EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView
EV180071 1/3 Homo sapiens Blood plasma SEC
UF
Brahmer A 2019 63%

Study summary

Full title
All authors
Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM.
Journal
J Extracell Vesicles
Abstract
Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide)
EV-METRIC
63% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC + UF
Protein markers
EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142
non-EV: / ApoA1
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Ultra filtration
Cut-off size (kDa)
30
Membrane type
Regenerated cellulose
Commercial kit
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD81/ CD9/ TSG101/ Syntenin/ CD41b/ CD63
Not detected EV-associated proteins
Sarcoglycan-alpha
Detected contaminants
ApoA1
Not detected contaminants
Detected EV-associated proteins
CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
106
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180050 3/6 Homo sapiens Cell culture supernatant dUC
Filtration
SEC
UF
Alice Gualerzi 2019 62%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
62% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: CD81/ Flotillin-1/ CD63
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
bone marrow-derived mesenchymal stem cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
> 0.45 µm,
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
2
Resin type
HiPrep 16/60 Sephacryl S-400 HR
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, Flotillin-1
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
Other
PMID previous EV particle analysis
Other
NTA
Report type
Mean
Reported size (nm)
247 ± 68
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV180050 4/6 Homo sapiens Cell culture supernatant dUC
Filtration
SEC
UF
Alice Gualerzi 2019 62%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
62% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
liver stem cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
> 0.45 µm,
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
2
Resin type
HiPrep 16/60 Sephacryl S-400 HR
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
Other
PMID previous EV particle analysis
Other
NTA
Report type
Mean
Reported size (nm)
228 ± 50
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV190020 1/3 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Kyuno, Daisuke 2019 57%

Study summary

Full title
All authors
Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M.
Journal
Int J Cancer
Abstract
Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (hide)
EV-METRIC
57% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Protein markers
EV:
non-EV:
Proteomics
yes
EV density (g/ml)
1.15-1.56
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
SW948
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1.28
Fraction processing
Centrifugation
Pelleting: volume per fraction
50
Pelleting: duration (min)
150
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
50
Pelleting-wash: duration (min)
150
Pelleting-wash: speed (g)
Type 45 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
NA
EV190020 2/3 Rattus norvegicus Cell culture supernatant DG
dUC
Filtration
Kyuno, Daisuke 2019 57%

Study summary

Full title
All authors
Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M.
Journal
Int J Cancer
Abstract
Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (hide)
EV-METRIC
57% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Protein markers
EV:
non-EV:
Proteomics
yes
EV density (g/ml)
1.15-1.56
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
ASML
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1.28
Fraction processing
Centrifugation
Pelleting: volume per fraction
50
Pelleting: duration (min)
150
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
50
Pelleting-wash: duration (min)
150
Pelleting-wash: speed (g)
Type 45 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
NA
EV180072 1/4 Homo sapiens Cell culture supernatant dUC
Total Exosome Isolation
UF
Fricke F 2019 57%

Study summary

Full title
All authors
Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J.
Journal
Int J Oncol
Abstract
In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide)
EV-METRIC
57% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Total Exosome Isolation + UF
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HCT116-TGFBR2
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting: time(min)
60
Pelleting: rotor type
ClickSeal Biocontainment Rotor with Lid; 24 x 1.5/2.0 mL Tubes
Pelleting: speed (g)
21100
Ultra filtration
Cut-off size (kDa)
10000
Membrane type
Polyethersulfone (PES)
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
Characterization: Particle analysis
NA
NTA
Report type
Median
Reported size (nm)
128
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV180072 2/4 Homo sapiens Cell culture supernatant dUC
Total Exosome Isolation
UF
Fricke F 2019 57%

Study summary

Full title
All authors
Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J.
Journal
Int J Oncol
Abstract
In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide)
EV-METRIC
57% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
genetically modified cell line
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Total Exosome Isolation + UF
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
genetically modified cell line
EV-producing cells
HCT116-TGFBR2
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting: time(min)
60
Pelleting: rotor type
ClickSeal Biocontainment Rotor with Lid Thermo Scientific
Pelleting: speed (g)
21100
Ultra filtration
Cut-off size (kDa)
10000
Membrane type
Polyethersulfone (PES)
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
Characterization: Particle analysis
NA
NTA
Report type
Median
Reported size (nm)
125
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV180060 1/2 Homo sapiens NA dUC
SEC
UF
Benedikter BJ 2019 57%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM
Journal
J Extracell Vesicles
Abstract
Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide)
EV-METRIC
57% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
Control condition
Focus vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + SEC + UF
Protein markers
EV: CD63/ MFGE8/ CD81/ TF/ HSP70/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
NA
Sample Condition
Control condition
EV-producing cells
BEAS-2B
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSP70/ CD81/ MFGE8/ CD63
Flow cytometry specific beads
Detected EV-associated proteins
TF/ CD63/ CD81/ CD9
Proteomics database
Yes
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NA
TRPS
Report type
Size range/distribution
Reported size (nm)
80-250
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
70
EV180060 2/2 Homo sapiens NA dUC
SEC
UF
Benedikter BJ 2019 57%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM
Journal
J Extracell Vesicles
Abstract
Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide)
EV-METRIC
57% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
1% cigarette smoke extract
Focus vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + SEC + UF
Protein markers
EV: TF/ CD81/ CD63/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
NA
Sample Condition
1% cigarette smoke extract
EV-producing cells
BEAS-2B
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Flow cytometry specific beads
Detected EV-associated proteins
TF/ CD63/ CD81/ CD9
Proteomics database
Yes
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NA
TRPS
Report type
Size range/distribution
Reported size (nm)
80-250
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
65
EV180009 3/3 Danio rerio Dissociated embryo dUC
IAF
Frederik J.Verweij 2019 57%

Study summary

Full title
All authors
Frederik J.Verweij, Celine Revenu, Guillaume Arras, Florent Dingli, Damarys Loew, Michiel D.Pegtel, Gautier Follain, Guillaume Allio, Jacky G.Goetz, Pascale Zimmermann, Philippe Herbomel, Filippo Del Bene, GraçaRaposo, Guillaumevan Niel
Journal
Cell Press
Abstract
Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physio (show more...)Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are captured, endocytosed, and degraded by patrolling macrophages and endothelial cells in the caudal vein plexus (CVP) in a scavenger receptor- and dynamin-dependent manner. Interference with exosome biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes. (hide)
EV-METRIC
57% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Dissociated embryo
Sample origin
Overexpression of CD63-phluorin in yolk syncitial layer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + IAF
Adj. k-factor
41.45 (pelleting) / 41.45 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Biogenesis/cargo sorting, Mechanism of uptake/transfer, New methodological development, Identification of content (omics approaches), Interorgan transfer of EVs in vivo
Sample
Species
Danio rerio
Sample Type
Dissociated embryo
Sample Condition
Overexpression of CD63-phluorin in yolk syncitial layer
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
TLA-120.1
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
41.45
Wash: time (min)
60
Wash: Rotor Type
TLA-120.2
Wash: speed (g)
100000
Wash: adjusted k-factor
41.45
Characterization: Protein analysis
Proteomics database
No
Characterization: Particle analysis
Nanoparticle tracking analysis
PMID previous EV particle analysis
Nanoparticle tracking analysis
NTA
Report type
Modus
Reported size (nm)
108
EV concentration
Yes
Particle yield
860000000000
EM
EM-type
Immune-EM
Proteïns
GFP
Image type
Close-up, Wide-field
Report size (nm)
60-200
Extra information
We have developed live cell imaging method to visualize and quantify exosome release (Verweij et al., JCB 2018). This method could be added to EV-track, e.g. as a measure to positively identify the endosomal origin of an EV population.
EV180082 10/10 Danio rerio Cell culture supernatant dUC Hyenne V 2019 57%

Study summary

Full title
All authors
Hyenne V, Ghoroghi S, Collot M, Bons J, Follain G, Harlepp S, Mary B, Bauer J, Mercier L, Busnelli I, Lefebvre O, Fekonja N, Garcia-Leon MJ, Machado P, Delalande F, López AA, Silva SG, Verweij FJ, van Niel G, Djouad F, Peinado H, Carapito C, Klymchenko AS, Goetz JG.
Journal
Dev cell
Abstract
Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly (show more...)Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo. (hide)
EV-METRIC
57% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Danio rerio
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Zmel1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
150
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
90
EV190080 1/3 Homo sapiens Cell culture supernatant DG
dUC
SEC
Size-exclusion chromatography (non-commercial)
Zaborowski MP 2019 56%

Study summary

Full title
All authors
Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, Zappulli V, Maas SLN, Allen RM, Rumde P, György B, Aufiero M, Schweiger MW, Lai CP, Weissleder R, Lee H, Vickers KC, Tannous BA, Breakefield XO.
Journal
Sci Rep
Abstract
Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter (show more...)Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + SEC + Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD63
non-EV:
Proteomics
no
EV density (g/ml)
1.11
Show all info
Study aim
New methodological development/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
OVCAR5
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120101
Wash: volume per pellet (ml)
40
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
120101
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
8%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
200620
Duration (min)
38
Fraction volume (mL)
350
Fraction processing
None
Size-exclusion chromatography
Total column volume (mL)
24
Sample volume/column (mL)
1
Resin type
Superdex 200
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63
Other 1
https://www.ncbi.nlm.nih.gov/pubmed/30943406
NA
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
50-120
Other particle analysis name(1)
Report type
EV-concentration
EV190049 1/3 Homo sapiens Cell culture supernatant dUC
qEV
Bliss CM 2019 56%

Study summary

Full title
All authors
Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L.
Journal
Matters
Abstract
Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD81/ Alix/ CD63/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Antigen targeting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NA
NTA
Report type
Not Reported
EV190049 2/3 Homo sapiens Cell culture supernatant dUC
qEV
Bliss CM 2019 56%

Study summary

Full title
All authors
Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L.
Journal
Matters
Abstract
Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Transfected with plasmid expressing EGFP
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD81/ Alix/ CD63/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Antigen targeting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Transfected with plasmid expressing EGFP
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NA
NTA
Report type
Modus
Reported size (nm)
161
EV concentration
Yes
EV190049 3/3 Homo sapiens Cell culture supernatant dUC
qEV
Bliss CM 2019 56%

Study summary

Full title
All authors
Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L.
Journal
Matters
Abstract
Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Transfected with plasmid expressing EGFP_C1C2
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD81/ Alix/ CD63/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Antigen targeting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Transfected with plasmid expressing EGFP_C1C2
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NA
NTA
Report type
Modus
Reported size (nm)
157
EV concentration
Yes
EV190027 1/1 Mus musculus Cell culture supernatant dUC Auber M 2019 56%

Study summary

Full title
All authors
Auber M, Fröhlich D, Drechsel O, Karaulanov E, Krämer-Albers EM.
Journal
J Extracell Vesicles
Abstract
Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from (show more...)Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions includes miRNA contaminants carried into the sample by defined media components. Subjecting unconditioned, EV-free medium to differential centrifugation followed by reverse transcription quantitative PCR (RT-qPCR) on RNA isolated from the pellet resulted in detection of miRNAs that had been classified as EV-enriched by RNA-seq or RT-qPCR of an isolated EV-fraction. Ribonuclease (RNase-A) and detergent treatment removed most but not all of the contaminating miRNAs. Further analysis of the defined media constituents identified Catalase as a main source of miRNAs co-isolating together with EVs. Hence, miRNA contaminants can be carried into EV-samples even under serum-free harvesting conditions using culture media that are expected to be chemically defined. Formulation of miRNA-free media supplements may provide a solution to collect EVs clean from confounding miRNAs, which however still remains a challenging task. Differential analysis of EVs collected under full medium and supplement-deprived conditions appears to provide a strategy to discriminate confounding and EV-associated RNA. In conclusion, we recommend careful re-evaluation and validation of EV small RNA-seq and RT-qPCR datasets by determining potential medium background. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary oligodendrocytes
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
103000
Characterization: Protein analysis
PMID previous EV protein analysis
Characterization: Particle analysis
NA
EV190026 1/2 Homo sapiens Serum dUC
qEV
Gualerzi A 2019 56%

Study summary

Full title
All authors
Gualerzi A, Picciolini S, Carlomagno C, Terenzi F, Ramat S, Sorbi S, Bedoni M.
Journal
Nanomedicine
Abstract
Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clin (show more...)Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clinical heterogeneity. Extracellular vesicles (EVs) were proposed as new biomarkers for PD because of their role as vehicles of multiple PD related molecules, but technical limitations exist in their detection and characterization in a clinical environment. We propose herein a Raman based protocol for the label-free analysis of circulating EVs as diagnostic and predictive tool for PD. After purification from serum of PD patients and healthy subjects, EVs were analyzed by Raman spectroscopy demonstrating the feasibility and reproducibility of the proposed biophotonic approach, its moderate accuracy in distinguishing PD patients from controls by their EV profile and the correlation between Raman data and clinical scales. Once validated, the Raman spectroscopy of circulating EVs could represent a reliable, automatable and sensitive method for the stratification of PD patients and for the evaluation of the effectiveness of rehabilitation and pharmacological treatments. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD63/ Flotillin1/ alpha-synuclein
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ alpha-synuclein
Detected contaminants
Albumin
NA
EM
EM-type
Transmission-EM
Image type
Close-up
EV190026 2/2 Homo sapiens Serum dUC
qEV
Gualerzi A 2019 56%

Study summary

Full title
All authors
Gualerzi A, Picciolini S, Carlomagno C, Terenzi F, Ramat S, Sorbi S, Bedoni M.
Journal
Nanomedicine
Abstract
Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clin (show more...)Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clinical heterogeneity. Extracellular vesicles (EVs) were proposed as new biomarkers for PD because of their role as vehicles of multiple PD related molecules, but technical limitations exist in their detection and characterization in a clinical environment. We propose herein a Raman based protocol for the label-free analysis of circulating EVs as diagnostic and predictive tool for PD. After purification from serum of PD patients and healthy subjects, EVs were analyzed by Raman spectroscopy demonstrating the feasibility and reproducibility of the proposed biophotonic approach, its moderate accuracy in distinguishing PD patients from controls by their EV profile and the correlation between Raman data and clinical scales. Once validated, the Raman spectroscopy of circulating EVs could represent a reliable, automatable and sensitive method for the stratification of PD patients and for the evaluation of the effectiveness of rehabilitation and pharmacological treatments. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD63/ Flotillin1/ alpha-synuclein
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Parkinson's disease
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ alpha-synuclein
Detected contaminants
Albumin
NA
EM
EM-type
Transmission-EM
Image type
Close-up
EV180082 6/10 Mus musculus Cell culture supernatant DG
dUC
Hyenne V 2019 56%

Study summary

Full title
All authors
Hyenne V, Ghoroghi S, Collot M, Bons J, Follain G, Harlepp S, Mary B, Bauer J, Mercier L, Busnelli I, Lefebvre O, Fekonja N, Garcia-Leon MJ, Machado P, Delalande F, López AA, Silva SG, Verweij FJ, van Niel G, Djouad F, Peinado H, Carapito C, Klymchenko AS, Goetz JG.
Journal
Dev cell
Abstract
Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly (show more...)Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Protein markers
EV: Alix/ TSG101
non-EV:
Proteomics
yes
EV density (g/ml)
1.14
Show all info
Study aim
Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
4T1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 28
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
3
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ TSG101
Proteomics database
Yes
NA
EV200045 3/5 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Filtration
Ultrafiltration
Ludwig Nils 2019 50%

Study summary

Full title
All authors
Ludwig N, Hong CS, Ludwig S, Azambuja JH, Sharma P, Theodoraki MN, Whiteside TL.
Journal
Current Protocols in Immunology
Abstract
A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presen (show more...)A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presented. Tumor-derived exosomes (TEX) are defined as a subset of extracellular vesicles (EVs) sized at 30 to 150 nm and originating from multivesicular bodies (MVBs). The method utilizes size exclusion chromatography (SEC) for recovery of exosomes from cell-line supernatants or cancer patients' plasma. The recovered exosomes are morphologically intact, aggregate-free, and functionally competent. Their molecular content parallels that of the parent tumor cells and they carry various immunoregulatory ligands known to modulate functions of immune cells. All exosomes isolated from tumor cell lines are TEX, while those isolated from plasma of cancer patients have to be fractionated into TEX and non-TEX. Mini-SEC allows for exosome isolation and recovery in quantities sufficient for molecular profiling, functional studies, and, in the case of plasma, further fractionation into TEX and non-TEX. The mini-SEC method can also be used for comparative studies of the exosome content in serial specimens of cancer patients' body fluids. (hide)
EV-METRIC
50% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial) + Filtration + Ultrafiltration
Protein markers
EV: None
non-EV:
Proteomics
no
Show all info
Study aim
Function/New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PCI-13
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
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
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
PMID previous EV protein analysis
PMID 30042174 + PMID 30370252
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
PMID 30042174 + PMID 30370252
Characterization: Particle analysis
NA
TRPS
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190100 1/10 Homo sapiens Cell culture supernatant ExoQuick Liao C 2019 50%

Study summary

Full title
All authors
Liao C, Zhou Q1, Zhang Z, Wu X, Zhou Z, Li B, Peng J, Shen L, Li D, Luo X, Yang L.
Journal
Cancer Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (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
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no