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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
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Experiment number
  • Experiments differ in Sample type, Isolation method
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Experiment number
  • Experiments differ in Sample type, Vesicle type, Culture condition
Experiment number
  • Experiments differ in Sample type, Vesicle type, Culture condition
Experiment number
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Experiment number
  • Experiments differ in Sample type, Vesicle type, Culture condition
Experiment number
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Experiment number
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Experiment number
  • Experiments differ in Sample type, Isolation method
Experiment number
  • Experiments differ in Sample type, Isolation method
Experiment number
  • Experiments differ in Sample type, Sample condition
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV190011 4/5 Mus musculus Tissue (Differential) (ultra)centrifugation
Density gradient
Cianciaruso C 2019 100%

Study summary

Full title
All authors
Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M
Journal
Cell Rep
Abstract
Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide)
EV-METRIC
100% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Tissue
Sample origin
MC38 tumor
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Density gradient
Protein markers
EV: Alix/ CD9/ CD63/ TSG101/ CD81/ GAPDH/ MRC1/ CD68/ actin-beta/ HER2/ CD11b/ TBXAS1/ COX1
non-EV: Calnexin/ Gp96
Proteomics
yes
EV density (g/ml)
1.14
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Tissue
Sample Condition
MC38 tumor
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
134,000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Continuous
Lowest density fraction
17%
Highest density fraction
78%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
35
Pelleting: duration (min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
134000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101/ CD81/ GAPDH/ MRC1/ CD68/ actin-beta/ HER2/ TBXAS1/ COX1
Detected contaminants
Calnexin/ Gp96
Flow cytometry specific beads
Detected EV-associated proteins
CD11b/ CD9
Flow cytometry
Type of Flow cytometry
Attune NxT apparatus
Hardware adjustments
Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep
Detected EV-associated proteins
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
190
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190011 5/5 Mus musculus Tissue (Differential) (ultra)centrifugation
Density gradient
Cianciaruso C 2019 100%

Study summary

Full title
All authors
Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M
Journal
Cell Rep
Abstract
Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide)
EV-METRIC
100% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Tissue
Sample origin
E0771 tumor
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Density gradient
Protein markers
EV: CD9/ MRC1/ COX1/ TBXAS1
non-EV: Gp96
Proteomics
yes
EV density (g/ml)
1.14
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Tissue
Sample Condition
E0771 tumor
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
134,000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
17%
Highest density fraction
77%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
35
Pelleting: duration (min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
134000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ MRC1/ COX1/ TBXAS1
Detected contaminants
Gp96
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
180
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV180011 1/1 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Kathrin Gärtner 2019 100%

Study summary

Full title
All authors
Kathrin Gärtner, Manja Luckner, Gerhard Wanner, Reinhard Zeidler
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are important mediators of cell–cell communication. Intriguingly, EVs (show more...)Extracellular vesicles (EVs) are important mediators of cell–cell communication. Intriguingly, EVs can be engineered and thus exploited for the targeted transfer of functional proteins of interest. Thus, engineered EVs may constitute attractive tools for the development of novel therapeutic interventions, like cancer immunotherapies, vaccinations or targeted drug delivery. Here, we describe a novel experimental immunotherapeutic approach for the adjuvant treatment of chronic lymphocytic leukaemia (CLL) based on engineered EVs carrying gp350, the major glycoprotein of Epstein–Barr virus (EBV), CD40L, a central immune accessory molecule and pp65, an immunodominant antigen of the human cytomegalovirus (CMV). We show that these engineered EVs specifically interact with malignant B cells from CLL patients and render these cells immunogenic to allogeneic and autologous EBV- and CMV-specific CD4+ and CD8+ T cells. Collectively, co-opting engineered EVs to re-target the strong herpesviral immunity in CLL patients to malignant cells constitutes an attractive strategy for the adjuvant treatment of a still incurable disease. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Overexpressing gp350, CD40L and/or pp65
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: Alix/ CD63/ TSG101
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Overexpressing gp350, CD40L and/or pp65
EV-producing cells
HEK293
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
2
Lowest density fraction
0.3
Highest density fraction
0.44
Sample volume (mL)
0.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 60 Ti
Speed (g)
160000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
30
Pelleting: duration (min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, TSG101
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.01
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-300
EV concentration
Yes
EM
EM-type
Transmission-EM/ Immune-EM
Image type
Close-up, Wide-field
EV190038 2/3 Homo sapiens Exudative seroma (Differential) (ultra)centrifugation
Density gradient
García-Silva S 2019 78%

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
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Exudative seroma
Sample origin
Melanoma patients stage III
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
Not specified
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Exudative seroma
Sample Condition
Melanoma patients stage III
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
8
Sample volume (mL)
0.1
Orientation
Top-down
Rotor type
Type 70.1 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
20
Pelleting: duration (min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ GAPDH/ CD81/ HSP90/ TRP2
Proteomics database
ProteomeXchange Consortium
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
148
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
63.6
EV190011 1/5 Mus musculus Cell culture supernatant (Differential) (ultra)centrifugation Cianciaruso C 2019 78%

Study summary

Full title
All authors
Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M
Journal
Cell Rep
Abstract
Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ vinculin/ GAPDH/ Alix/ TSG101/ Syntenin1/ actin-beta/ CD9/ CD63/ HER2/ CD11b
non-EV: Calnexin/ Gp96
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MC38
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
134,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD81/ vinculin/ GAPDH/ Alix/ TSG101/ Syntenin1/ actin-beta/ CD9/ CD63
Detected contaminants
Calnexin/ Gp96
Flow cytometry specific beads
Detected EV-associated proteins
HER2/ CD11b/ CD9
Flow cytometry
Type of Flow cytometry
Attune NxT apparatus
Hardware adjustments
Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep
Detected EV-associated proteins
HER2
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
150
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190011 3/5 Mus musculus Cell culture supernatant (Differential) (ultra)centrifugation Cianciaruso C 2019 78%

Study summary

Full title
All authors
Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M
Journal
Cell Rep
Abstract
Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ MRC1/ GAPDH/ CD63/ TSG101/ CD9/ CD68/ MRC1/ Alix
non-EV: Gp96
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Bone marrow-derived macrophages
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
134,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD81/ MRC1/ GAPDH/ CD63/ TSG101/ CD9/ CD68/ MRC1/ Alix
Not detected EV-associated proteins
Not detected contaminants
Gp96
Flow cytometry
Type of Flow cytometry
Attune NxT apparatus
Hardware adjustments
Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep
Detected EV-associated proteins
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
140
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV180021 3/4 Homo sapiens Serum dUC
Filtration
Bachurski, Daniel 2019 77%

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
77% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
209.7 (pelleting) / 89.2 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ 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
Serum
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
209.7
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Filtration steps
0.22µm or 0.2µm
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
NTA
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-100
Other particle analysis name(1)
ExoView
Report type
Size range/distribution
Report size
50-100
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 4/4 Homo sapiens Cell culture supernatant dUC
Filtration
Bachurski, Daniel 2019 77%

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
77% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
209.7 (pelleting) / 89.2 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ 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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
209.7
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Filtration steps
0.22µm or 0.2µm
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
NTA
Report type
Size range/distribution
Reported size (nm)
50-400
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
Other particle analysis name(1)
ExoView
Report type
Size range/distribution
Report size
50-100
EV-concentration
No
Extra information
EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView
EV190038 1/3 Homo sapiens Blood plasma (Differential) (ultra)centrifugation 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% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Melanoma patients stage III
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Melanoma patients stage III
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
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
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ Flotillin-1/ TSG101/ beta-actin
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Other
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: CD63/ CD81/ Flotillin-1
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Other
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: CD63/ CD81/ Flotillin-1
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Other
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ Flotillin-1/ TSG101/ beta-actin
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Other
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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)
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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)
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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)
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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)
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD9/ CD81/ TSG101/ CD29
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
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
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
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% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
892 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ 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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
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
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% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
892 (washing)
Protein markers
EV: CD63/ HSP70/ 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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
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
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
EV180050 3/6 Homo sapiens Cell culture supernatant dUC
Filtration
SEC
Ultrafiltration
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% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + Ultrafiltration
Protein markers
EV: CD63/ CD81/ Flotillin-1
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
Isolation Method
Differential ultracentrifugation
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
PMID previous EV particle analysis
Other
Extra particle analysis
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
Ultrafiltration
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% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + Ultrafiltration
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ Flotillin-1/ TSG101/ beta-actin
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
Isolation Method
Differential ultracentrifugation
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
PMID previous EV particle analysis
Other
Extra particle analysis
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 Density gradient
(Differential) (ultra)centrifugation
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% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + (Differential) (ultra)centrifugation + Filtration
Protein markers
EV: None
non-EV: None
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
Characterization: Particle analysis
EV190020 2/3 Rattus norvegicus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
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% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + (Differential) (ultra)centrifugation + Filtration
Protein markers
EV: None
non-EV: None
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
Characterization: Particle analysis
EV180060 1/NA Homo sapiens NA Filtration
Ultrafiltration
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
Control condition
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Filtration + Ultrafiltration + (Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
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
Isolation Method
Differential ultracentrifugation
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
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/NA Homo sapiens NA Filtration
Ultrafiltration
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
1% cigarette smoke extract
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Filtration + Ultrafiltration + (Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
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
Isolation Method
Differential ultracentrifugation
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
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
Immunoaffinity capture (non-commercial)
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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Dissociated embryo
Sample origin
Overexpression of CD63-phluorin in yolk syncitial layer
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Immunoaffinity capture (non-commercial)
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Particle analysis
PMID previous EV particle analysis
Nanoparticle tracking analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
108
EV concentration
Yes
Particle yield
860000000000
EM
EM-type
Immune-EM
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 (Differential) (ultra)centrifugation 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% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
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
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
90
EV180082 6/10 Mus musculus Cell culture supernatant (Differential) (ultra)centrifugation
Density gradient
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% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Density gradient
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
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 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
Characterization: Particle analysis
EV190011 2/5 Mus musculus Cell culture supernatant (Differential) (ultra)centrifugation Cianciaruso C 2019 44%

Study summary

Full title
All authors
Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M
Journal
Cell Rep
Abstract
Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide)
EV-METRIC
44% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ GAPDH/ CD81
non-EV:
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
E0771
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
134,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD9/ GAPDH
Not detected EV-associated proteins
CD81
Characterization: Particle analysis
EV170031 1/1 Homo sapiens Serum dUC
Filtration
Ramanathan S 2019 44%

Study summary

Full title
All authors
Ramanathan S, Douglas SR, Alexander GM, Shenoda BB, Barrett JE, Aradillas E, Sacan A, Ajit SK
Journal
J Transl Med
Abstract
BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employ (show more...)BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE. (hide)
EV-METRIC
44% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Complex regional pain syndrome
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
142.9 (pelleting) / 142.9 (washing)
Protein markers
EV: CD63/ CD81
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Complex regional pain syndrome
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
70
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Wash: adjusted k-factor
142.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
85.7
EV concentration
Yes
Particle yield
394000000000
EM
EM-type
Transmission-EM/ Immune-EM
Image type
Close-up, Wide-field
EV190020 3/3 Rattus norvegicus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Filtration
Kyuno, Daisuke 2019 43%

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
43% (77th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
claudin 7 knockdown
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + (Differential) (ultra)centrifugation + Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
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
claudin 7 knockdown
EV-producing cells
ASML
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
Protein Concentration Method
Bradford
Characterization: Particle analysis
EV190012 1/3 Mus musculus Cell culture supernatant (Differential) (ultra)centrifugation
Ultrafiltration
Density cushion
Filtration
Commercial method
Vu LT 2019 38%

Study summary

Full title
All authors
Vu LT, Peng B, Zhang DX, Ma V, Mathey-Andrews CA, Lam CK, Kiomourtzis T, Jin J, McReynolds L, Huang L, Grimson A, Cho WC, Lieberman J, Le MT.
Journal
J Extracell Vesicles
Abstract
Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions (show more...)Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts. (hide)
EV-METRIC
38% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Ultrafiltration + Density cushion + Filtration + Commercial method
Protein markers
EV: TSG101/ Alix
non-EV: Beta-Actin
Proteomics
no
Show all info
Study aim
Function
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
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Filtration steps
0.45µm > x > 0.22µm,
Ultra filtration
Cut-off size (kDa)
Not spec
Membrane type
Regenerated cellulose
Commercial kit
qEV
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
TSG101/ Alix
Not detected contaminants
Beta-Actin
Flow cytometry specific beads
Selected surface protein(s)
CD63
Detected EV-associated proteins
CD63
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
50 U
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
120
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180082 1/10 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Hyenne V 2019 29%

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
29% (63rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation
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
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
451-LU
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
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
Protein Concentration Method
BCA
Proteomics database
Yes
Characterization: Particle analysis
EV180082 2/10 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation Hyenne V 2019 29%

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
29% (63rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell cultu