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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200054 4/7 Mus musculus Brain tissue (d)(U)C
Filtration
DC
Huang, Yiyao 2020 67%

Study summary

Full title
All authors
Yiyao Huang, Lesley Cheng, Andrey Turchinovich, Vasiliki Mahairaki, Juan C Troncoso, Olga Pletniková, Norman J Haughey, Laura J Vella, Andrew F Hill, Lei Zheng, Kenneth W Witwer
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processe (show more...)Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease. (hide)
EV-METRIC
67% (83rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Brain tissue
Sample origin
Control condition
Focus vesicles
extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration + DC
Protein markers
EV: CD9/ TSG101/ CD81/ Syntenin
non-EV: GM130/ Calnexin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV- related methods
Sample
Species
Mus musculus
Sample Type
Brain tissue
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
AH-650
Pelleting: speed (g)
10000
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Not detected EV-associated proteins
CD81/ Syntenin
Detected contaminants
GM130/ Calnexin
Characterization: Particle analysis
NTA
EV concentration
Yes
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV200031 1/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
800
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
800
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
200-600
Report type
Not Reported
EV200031 2/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
45
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
16000
Wash: volume per pellet (ml)
1
Wash: time (min)
45
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
16000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
50-400
Report type
Not Reported
EV200031 3/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
30-200
Report type
Not Reported
EV200031 5/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
800
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
800
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
200-600
Report type
Not Reported
EV200031 6/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
45
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
16000
Wash: volume per pellet (ml)
1
Wash: time (min)
45
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
16000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
50-400
Report type
Not Reported
EV200031 7/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
30-200
Report type
Not Reported
EV200012 1/2 Rattus norvegicus Cell culture supernatant (d)(U)C Doreen Matthies 2020 67%

Study summary

Full title
All authors
Doreen Matthies, Nathanael Y J Lee, Ian Gatera, H Amalia Pasolli, Xiaowei Zhao, Hui Liu, Deepika Walpita, Zhe Liu, Zhiheng Yu, Maria S Ioannou
Journal
Sci Rep
Abstract
Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been imp (show more...)Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Primary cortical neurons
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: tubulin/ Flotillin1/ syntenin
non-EV: gp96
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Primary cortical neurons
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
300000
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ syntenin
Not detected EV-associated proteins
tubulin
Not detected contaminants
gp96
PMID previous EV particle analysis
Extra particle analysis
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
99.07+/-69.87
EV concentration
Yes
EV200000 3/4 Homo sapiens pleural effusion (d)(U)C Ping, Luo 2020 67%

Study summary

Full title
All authors
Ping Luo, Kaimin Mao, Juanjuan Xu, Feng Wu, Xuan Wang, Sufei Wang, Mei Zhou, Limin Duan, Qi Tan, Guangzhou Ma, Guanghai Yang, Ronghui Du, Hai Huang, Qi Huang, Yumei Li, Mengfei Guo, Yang Jin
Journal
J Extracell Vesicles
Abstract
Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of (show more...)Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of tuberculosis pleural effusion (TPE) and malignancy pleural effusion (MPE) remain challenging. Although extracellular vesicles (EVs) have been confirmed as promising sources of disease biomarkers, little is known about the metabolite compositions of its subpopulations and their roles in the diagnosis of pleural effusion. Here, we performed metabolomics and lipidomics analysis to investigate the metabolite characteristics of two EV subpopulations derived from pleural effusion by differential ultracentrifugation, namely large EVs (lEVs, pelleted at 20,000 × g) and small EVs (sEVs, pelleted at 110,000 × g), and assessed their metabolite differences between tuberculosis and malignancy. A total of 579 metabolites, including amino acids, acylcarnitines, organic acids, steroids, amides and various lipid species, were detected. The results showed that the metabolic profiles of lEVs and sEVs overlapped with and difference from each other but significantly differed from those of pleural effusion. Additionally, different type of vesicles and pleural effusion showed unique metabolic enrichments. Furthermore, lEVs displayed more significant and larger metabolic alterations between the tuberculosis and malignancy groups, and their differential metabolites were more closely related to clinical parameters than those of sEV. Finally, a panel of four biomarker candidates, including phenylalanine, leucine, phosphatidylcholine 35:0, and sphingomyelin 44:3, in pleural lEVs was defined based on the comprehensive discovery and validation workflow. This panel showed high performance for distinguishing TPE and MPE, particularly in patients with delayed or missed diagnosis, such as the area under the receiver-operating characteristic curve (AUC) >0.95 in both sets. We conducted comprehensive metabolic profiling analysis of EVs, and further explored the metabolic reprogramming of tuberculosis and malignancy at the level of metabolites in lEVs and sEVs, providing insight into the mechanism of pleural effusion, and identifying novel biomarkers for diagnosing TPE and MPE. (hide)
EV-METRIC
67% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
pleural effusion
Sample origin
lung cancer
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: CD81/ TSG101/ CD63/ CD9
non-EV: calnexin/ GM130
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
pleural effusion
Sample Condition
lung cancer
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
30
Wash: time (min)
90
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
110000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
200-1000 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected EV-associated proteins
CD81
Not detected contaminants
calnexin/ GM130
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
252.3
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 199903278
EM
EM-type
Transmission-EM
Image type
Close-up
EV200000 4/4 Homo sapiens pleural effusion (d)(U)C Ping, Luo 2020 67%

Study summary

Full title
All authors
Ping Luo, Kaimin Mao, Juanjuan Xu, Feng Wu, Xuan Wang, Sufei Wang, Mei Zhou, Limin Duan, Qi Tan, Guangzhou Ma, Guanghai Yang, Ronghui Du, Hai Huang, Qi Huang, Yumei Li, Mengfei Guo, Yang Jin
Journal
J Extracell Vesicles
Abstract
Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of (show more...)Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of tuberculosis pleural effusion (TPE) and malignancy pleural effusion (MPE) remain challenging. Although extracellular vesicles (EVs) have been confirmed as promising sources of disease biomarkers, little is known about the metabolite compositions of its subpopulations and their roles in the diagnosis of pleural effusion. Here, we performed metabolomics and lipidomics analysis to investigate the metabolite characteristics of two EV subpopulations derived from pleural effusion by differential ultracentrifugation, namely large EVs (lEVs, pelleted at 20,000 × g) and small EVs (sEVs, pelleted at 110,000 × g), and assessed their metabolite differences between tuberculosis and malignancy. A total of 579 metabolites, including amino acids, acylcarnitines, organic acids, steroids, amides and various lipid species, were detected. The results showed that the metabolic profiles of lEVs and sEVs overlapped with and difference from each other but significantly differed from those of pleural effusion. Additionally, different type of vesicles and pleural effusion showed unique metabolic enrichments. Furthermore, lEVs displayed more significant and larger metabolic alterations between the tuberculosis and malignancy groups, and their differential metabolites were more closely related to clinical parameters than those of sEV. Finally, a panel of four biomarker candidates, including phenylalanine, leucine, phosphatidylcholine 35:0, and sphingomyelin 44:3, in pleural lEVs was defined based on the comprehensive discovery and validation workflow. This panel showed high performance for distinguishing TPE and MPE, particularly in patients with delayed or missed diagnosis, such as the area under the receiver-operating characteristic curve (AUC) >0.95 in both sets. We conducted comprehensive metabolic profiling analysis of EVs, and further explored the metabolic reprogramming of tuberculosis and malignancy at the level of metabolites in lEVs and sEVs, providing insight into the mechanism of pleural effusion, and identifying novel biomarkers for diagnosing TPE and MPE. (hide)
EV-METRIC
67% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
pleural effusion
Sample origin
lung cancer
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD81/ CD63/ CD9
non-EV: calnexin/ GM130
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
pleural effusion
Sample Condition
lung cancer
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
30
Wash: time (min)
90
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
110000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
50-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected contaminants
calnexin/ GM130
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
136.4
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 168611013
EM
EM-type
Transmission-EM
Image type
Close-up
EV190069 1/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Mariscal, Javier 2020 67%

Study summary

Full title
All authors
Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PC3
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C
Protein markers
EV: CD81/ TSG101/ CD9/ HSPA5/ KRT18
non-EV:
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Biomarker/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 free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
30%
Total gradient volume, incl. sample (mL)
16.2
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 28
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
2.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
Other;Pierce 660nm
Western Blot
Detected EV-associated proteins
HSPA5/ KRT18
Not detected EV-associated proteins
CD81/ TSG101/ CD9
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
1770
EV concentration
Yes
EV190069 2/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Mariscal, Javier 2020 67%

Study summary

Full title
All authors
Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PC3
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C
Protein markers
EV: TSG101/ CD81/ CD9/ HSPA5/ KRT18
non-EV:
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Biomarker/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 free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
30%
Total gradient volume, incl. sample (mL)
16.2
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 28
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
2.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Other;Pierce 660nm
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ CD81
Not detected EV-associated proteins
HSPA5/ KRT18
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
131
EV concentration
Yes
EV190044 5/11 Homo sapiens Cell culture supernatant DG
(d)(U)C
Driedonks, Tom A.P. 2020 67%

Study summary

Full title
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary neutrophils
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C
Protein markers
EV: CD63/ CD9
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary neutrophils
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63
Not detected contaminants
Calnexin
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
EV concentration
Yes
EV190044 6/11 Homo sapiens Cell culture supernatant DG
(d)(U)C
Driedonks, Tom A.P. 2020 67%

Study summary

Full title
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary neutrophils
Sample origin
LPS stimulated
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C
Protein markers
EV: CD63/ CD9
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
LPS stimulated
EV-producing cells
primary neutrophils
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63
Not detected contaminants
Calnexin
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
EV180079 1/2 Mus musculus Cell culture supernatant (d)(U)C Lucia Paolini 2020 67%

Study summary

Full title
All authors
Lucia Paolini, Stefania Federici, Giovanni Consoli, Diletta Arceri, Annalisa Radeghieri, Ivano Alessandri, Paolo Bergese
Journal
J Extracell Vesicles
Abstract
Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the (show more...)Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the common strategy is based on searching and probing set of molecular components and physical properties intended to be univocally characteristics of the target subpopulation. Pitfalls include the risk to opt for an unsuitable marker set - which may either not represent the subpopulation or also cover other unintended subpopulations - and the need to use different characterization techniques and equipment. This approach focused on specific markers may result inadequate to routinely deal with EV subpopulations that have an intrinsic high level of heterogeneity. In this paper, we show that Fourier-transform Infrared (FT-IR) spectroscopy can provide a collective fingerprint of EV subpopulations in one single experiment. FT-IR measurements were performed on large (LEVs, ~600 nm), medium (MEVs, ~200 nm) and small (SEVs ~60 nm) EVs enriched from two different cell lines medium: murine prostate cancer (TRAMP-C2) and skin melanoma (B16). Spectral regions between 3100-2800 cm-1 and 1880-900 cm-1, corresponding to functional groups mainly ascribed to lipid and protein contributions, were acquired and processed by Principal Component Analysis (PCA). LEVs, MEVs and SEVs were separately grouped for both the considered cell lines. Moreover, subpopulations of the same size but from different sources were assigned (with different degrees of accuracy) to two different groups. These findings demonstrate that FT-IR has the potential to quickly fingerprint EV subpopulations as a whole, suggesting an appealing complement/alternative for their characterization and grading, extendable to healthy and pathological EVs and fully artificial nanovesicles. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
TRAMP-C2
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: ADAM10/ Annexin V/ Flotillin1/ CD81
non-EV: GM130
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
TRAMP-C2
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
240
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Colorimetric Nanoplasmonic Assay
Western Blot
Detected EV-associated proteins
Flotillin1/ Annexin V/ ADAM10/ CD81
Not detected contaminants
GM130
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
EM
EM-type
Atomic force-EM
Image type
Close-up, Wide-field
Report size (nm)
large EVs 570 nm, medium EVS 190 nm, Small EVs 70 nm
EV180079 2/2 Mus musculus Cell culture supernatant (d)(U)C Lucia Paolini 2020 67%

Study summary

Full title
All authors
Lucia Paolini, Stefania Federici, Giovanni Consoli, Diletta Arceri, Annalisa Radeghieri, Ivano Alessandri, Paolo Bergese
Journal
J Extracell Vesicles
Abstract
Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the (show more...)Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the common strategy is based on searching and probing set of molecular components and physical properties intended to be univocally characteristics of the target subpopulation. Pitfalls include the risk to opt for an unsuitable marker set - which may either not represent the subpopulation or also cover other unintended subpopulations - and the need to use different characterization techniques and equipment. This approach focused on specific markers may result inadequate to routinely deal with EV subpopulations that have an intrinsic high level of heterogeneity. In this paper, we show that Fourier-transform Infrared (FT-IR) spectroscopy can provide a collective fingerprint of EV subpopulations in one single experiment. FT-IR measurements were performed on large (LEVs, ~600 nm), medium (MEVs, ~200 nm) and small (SEVs ~60 nm) EVs enriched from two different cell lines medium: murine prostate cancer (TRAMP-C2) and skin melanoma (B16). Spectral regions between 3100-2800 cm-1 and 1880-900 cm-1, corresponding to functional groups mainly ascribed to lipid and protein contributions, were acquired and processed by Principal Component Analysis (PCA). LEVs, MEVs and SEVs were separately grouped for both the considered cell lines. Moreover, subpopulations of the same size but from different sources were assigned (with different degrees of accuracy) to two different groups. These findings demonstrate that FT-IR has the potential to quickly fingerprint EV subpopulations as a whole, suggesting an appealing complement/alternative for their characterization and grading, extendable to healthy and pathological EVs and fully artificial nanovesicles. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
B16
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: ADAM10/ Annexin V/ Flotillin1/ CD81
non-EV: GM130
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
B16
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
240
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Colorimetric Nanoplasmonic assay
Western Blot
Detected EV-associated proteins
Flotillin1/ Annexin V/ ADAM10/ CD81
Not detected contaminants
GM130
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
EM
EM-type
Atomic force-EM
Image type
Close-up, Wide-field
Report size (nm)
large EVS 690 nm, medium Evs 230 nm, small Evs 50 nm
EV180057 1/1 Rattus norvegicus Cell culture supernatant (d)(U)C Gissi, Clarissa 2020 67%

Study summary

Full title
All authors
Clarissa Gissi, Annalisa Radeghieri, Cristina Antonetti Lamorgese Passeri, Marialucia Gallorini, Lucia Calciano, Francesco Oliva, Francesca Veronesi, Andrea Zendrini, Amelia Cataldi, Paolo Bergese, Nicola Maffulli, Anna Concetta Berardi
Journal
PLoS One
Abstract
Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely med (show more...)Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely mediated through paracrine actions. Currently, extracellular vesicles (EVs) released by MSCs are major mediators of these paracrine effects. We evaluated whether rat-bone-marrow-MSC-derived EVs (rBMSCs-EVs) can ameliorate tendon injury in an in vivo rat model. Pro-collagen1A2 and MMP14 protein are expressed in rBMSC-EVs, and are important factors for extracellular-matrix tendon-remodeling. In addition, we found pro-collagen1A2 in rBMSC-EV surface-membranes by dot blot. In vitro on cells isolated from Achilles tendons, utilized as rBMSC -EVs recipient cells, EVs at both low and high doses induce migration of tenocytes; at higher concentration, they induce proliferation and increase expression of Collagen type I in tenocytes. Pretreatment with trypsin abrogate the effect of EVs on cell proliferation and migration, and the expression of collagen I. When either low- or high-dose rBMSCs-EVs were injected into a rat-Achilles tendon injury-model (immediately after damage), at 30 days, rBMSC-EVs were found to have accelerated the remodeling stage of tendon repair in a dose-dependent manner. At histology and histomorphology evaluation, high doses of rBMSCs-EVs produced better restoration of tendon architecture, with optimal tendon-fiber alignment and lower vascularity. Higher EV-concentrations demonstrated greater expression of collagen type I and lower expression of collagen type III. BMSC-EVs hold promise as a novel cell-free modality for the management of tendon injuries. (hide)
EV-METRIC
67% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
rBMSC
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: CD81/ Pro-collagen1A2/ Annexin/ TERT/ Annexin V
non-EV: Argonaute2/ GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
rBMSC
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD81/ Annexin V/ TERT/ Pro-collagen1A2
Detected contaminants
Argonaute2
Not detected contaminants
GM130
Characterization: Particle analysis
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
95
Report type
Not Reported
EV-concentration
Yes
Particle yield
1E11 per particles mL starting sample
XL5296IL 1/2 Macaca mulatta Cervicovaginal lavage fluid (d)(U)C Zhao, Zezhou 2020 66%

Study summary

Full title
All authors
Zezhou Zhao, Dillon C Muth, Kathleen Mulka, Zhaohao Liao, Bonita H Powell, Grace V Hancock, Kelly A Metcalf Pate, Kenneth W Witwer
Journal
FEBS Open Bio
Abstract
Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage ( (show more...)Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract. (hide)
EV-METRIC
66% (66th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cervicovaginal lavage fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Adj. k-factor
113.6 (pelleting)
Protein markers
EV: CD81/ CD63
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Macaca mulatta
Sample Type
Cervicovaginal lavage fluid
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
AH-650
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
113.6
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81
Not detected contaminants
calnexin
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
xx-xxx
EV concentration
Yes
Particle yield
2.5E7-3.5E9
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
up to 200
Extra information
Sample volumes and yield limited the isolation and characterization steps we were able to perform.
XL5296IL 2/2 Macaca mulatta Cervicovaginal lavage fluid (d)(U)C Zhao, Zezhou 2020 66%

Study summary

Full title
All authors
Zezhou Zhao, Dillon C Muth, Kathleen Mulka, Zhaohao Liao, Bonita H Powell, Grace V Hancock, Kelly A Metcalf Pate, Kenneth W Witwer
Journal
FEBS Open Bio
Abstract
Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage ( (show more...)Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract. (hide)
EV-METRIC
66% (66th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cervicovaginal lavage fluid
Sample origin
SIVmac251-infected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Adj. k-factor
113.6 (pelleting)
Protein markers
EV: CD81/ CD63
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Macaca mulatta
Sample Type
Cervicovaginal lavage fluid
Sample Condition
SIVmac251-infected
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
AH-650
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
113.6
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81
Not detected contaminants
calnexin
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
xx-xxx
EV concentration
Yes
Particle yield
1.75E8-2.15E9
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
up to 200 nm
Extra information
Samples from SIV Infected animals were pooled for WB analysis
EV200038 4/4 Homo sapiens Blood plasma CD31 MicroBead Kit Prattichizzo, Francesco 2020 63%

Study summary

Full title
All authors
Francesco Prattichizzo, Valeria De Nigris, Jacopo Sabbatinelli, Angelica Giuliani, Carlos Castaño, Marcelina Párrizas, Isabel Crespo, Annalisa Grimaldi, Nicolò Baranzini, Rosangela Spiga, Elettra Mancuso, Maria Rita Rippo, Antonio Domenico Procopio, Anna Novials, Anna Rita Bonfigli, Silvia Garavelli, Lucia La Sala, Giuseppe Matarese, Paola de Candia, Fabiola Olivieri, Antonio Ceriello
Journal
Diabetes
Abstract
Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (show more...)Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (T2DM). Blood circulating miRNAs have been proposed as a potential tool to detect T2DM complications, but the lack of tissue specificity, among other reasons, has hampered their translation to clinical settings. Extracellular vesicle (EV)-shuttled miRNAs have been proposed as an alternative approach. Here, we adapted an immunomagnetic bead-based method to isolate plasma CD31+ EVs to harvest vesicles deriving from tissues relevant for T2DM complications. Surface marker characterization showed that CD31+ EVs were also positive for a range of markers typical of both platelets and activated endothelial cells. After characterization, we quantified 11 candidate miRNAs associated with vascular performance and shuttled by CD31+ EVs in a large (n = 218) cross-sectional cohort of patients categorized as having T2DM without complications, having T2DM with complications, and control subjects. We found that 10 of the tested miRNAs are affected by T2DM, while the signature composed by miR-146a, -320a, -422a, and -451a efficiently identified T2DM patients with complications. Furthermore, another CD31+ EV-shuttled miRNA signature, i.e., miR-155, -320a, -342-3p, -376, and -422a, detected T2DM patients with a previous major adverse cardiovascular event. Many of these miRNAs significantly correlate with clinical variables held to play a key role in the development of complications. In addition, we show that CD31+ EVs from patients with T2DM are able to promote the expression of selected inflammatory mRNAs, i.e., CCL2, IL-1α, and TNFα, when administered to endothelial cells in vitro. Overall, these data suggest that the miRNA cargo of plasma CD31+ EVs is largely affected by T2DM and related complications, encouraging further research to explore the diagnostic potential and the functional role of these alterations. (hide)
EV-METRIC
63% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Type 2 Diabetes
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
CD31 MicroBead Kit
Protein markers
EV: TSG101/ CD63/ CD81/ CD31/ CD9/ Alix
non-EV: ApoA1
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Type 2 Diabetes
Separation Method
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ CD31/ CD63/ TSG101
Not detected contaminants
ApoA1
Flow cytometry specific beads
Detected EV-associated proteins
CD31/ CD9/ CD63/ CD81
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
120
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200042 2/2 Mus musculus Cell culture supernatant DG
(d)(U)C
DC
Filtration
Laura Bouchareychas 2020 63%

Study summary

Full title
All authors
Laura Bouchareychas, Phat Duong, Sergio Covarrubias, Eric Alsop, Tuan Anh Phu, Allen Chung, Michael Gomes, David Wong, Bessie Meechoovet, Allyson Capili, Ryo Yamamoto, Hiromitsu Nakauchi, Michael T McManus, Susan Carpenter, Kendall Van Keuren-Jensen, Robert L Raffai
Journal
Cell Rep
Abstract
Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remai (show more...)Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery. (hide)
EV-METRIC
63% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Immortalized bone marrow-derived macrophages (iBMDMs)
Sample origin
genetically modified cell line
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + DC + Filtration
Protein markers
EV: Alix/ Flotillin1
non-EV: Calnexin/ GM130
Proteomics
no
EV density (g/ml)
1.09
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
genetically modified cell line
EV-producing cells
Immortalized bone marrow-derived macrophages (iBMDMs)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Density gradient
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)
12
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Iodixanol
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix
Not detected contaminants
Calnexin/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
Other;RNase A/T1 Mix
RNAse concentration
0.4
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
74.58
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 2.40E+09
EV200037 1/2 Homo sapiens Cell culture supernatant Density gradient
Size-exclusion chromatography (non-commercial)
PEG precipitation
Xiaogang Zhang 2020 63%

Study summary

Full title
All authors
Xiaogang Zhang , Ellen G. F. Borg , A. Manuel Liaci , Harmjan R. Vos & Willem Stoorvogel
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellul (show more...)Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment. (hide)
EV-METRIC
63% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HLA-DR15+ B cells
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + Size-exclusion chromatography (non-commercial) + PEG precipitation
Protein markers
EV: CD81/ MHC2/ CD63
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.13
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HLA-DR15+ B cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Density gradient
Density medium
Other medium;iohexol
Type
Continuous
Lowest density fraction
0%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 60 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
0.3
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.6
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ MHC2/ CD81
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200037 2/2 Homo sapiens Blood plasma Density gradient
Size-exclusion chromatography (non-commercial)
PEG precipitation
Xiaogang Zhang 2020 63%

Study summary

Full title
All authors
Xiaogang Zhang , Ellen G. F. Borg , A. Manuel Liaci , Harmjan R. Vos & Willem Stoorvogel
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellul (show more...)Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment. (hide)
EV-METRIC
63% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + Size-exclusion chromatography (non-commercial) + PEG precipitation
Protein markers
EV: CD81/ CD63/ CD9
non-EV: None
Proteomics
yes
EV density (g/ml)
1.09-1.13
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Density gradient
Density medium
Other medium;iohexol
Type
Continuous
Lowest density fraction
0%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 60 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
0.3
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.6
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ CD81
Proteomics
Proteomics database
Yes:
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Wide-field
EV190050 1/1 Mus musculus Cell culture supernatant DG
Filtration
SEC
Size-exclusion chromatography (non-commercial)
Ger J A Arkesteijn 2020 63%

Study summary

Full title
All authors
Ger J A Arkesteijn, Estefanía Lozano-Andrés, Sten F W M Libregts, Marca H M Wauben
Journal
Cytometry A
Abstract
Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of (show more...) Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron-sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV-related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV-subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC-detection of submicron-sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron-sized particles. We found that a combination of an 8-mm obscuration bar and a 200-μm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100-nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron-sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron-sized particles. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. (hide)
EV-METRIC
63% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
4T1
Sample origin
breast tumor model
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + Filtration + SEC + Size-exclusion chromatography (non-commercial)
Protein markers
EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ Flotillin2/ HSP70/ MHC2/ CD9/ MHC1
non-EV:
Proteomics
no
EV density (g/ml)
1.10-1.12
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
breast tumor model
EV-producing cells
4T1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Density gradient
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)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1091
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45µm > x > 0.22µm,
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63
Not detected EV-associated proteins
HSP90/ HSP70/ MHC1/ CD81/ Flotillin1/ TSG101/ MHC2/ Flotillin2/ Alix
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
139.8
EV concentration
Yes
EV190036 1/3 Homo sapiens Cell culture supernatant Filtration
qEV
Hicks, David 2020 63%

Study summary

Full title
All authors
David A Hicks, Alys C Jones, Nicola J Corbett, Kate Fisher, Stuart M Pickering-Brown, Mark P Ashe, Nigel M Hooper
Journal
Neurochem Res.
Abstract
Healthy brain function is mediated by several complementary signalling pathways, many of which are d (show more...)Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30–100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons. (hide)
EV-METRIC
63% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
OX1-19
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Filtration + qEV
Protein markers
EV: TSG101/ CD9
non-EV: Mitofilin/ Grp78
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
OX1-19
EV-harvesting Medium
Serum free medium
Separation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Not detected contaminants
Mitofilin/ Grp78
Proteomics
Proteomics database
No
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.05
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
SEC fraction-dependent
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190036 2/3 Homo sapiens Cell culture supernatant Filtration
qEV
Hicks, David 2020 63%

Study summary

Full title
All authors
David A Hicks, Alys C Jones, Nicola J Corbett, Kate Fisher, Stuart M Pickering-Brown, Mark P Ashe, Nigel M Hooper
Journal
Neurochem Res.
Abstract
Healthy brain function is mediated by several complementary signalling pathways, many of which are d (show more...)Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30–100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons. (hide)
EV-METRIC
63% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
OX1-19
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Filtration + qEV
Protein markers
EV: TSG101/ CD9
non-EV: Mitofilin/ Grp78
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
OX1-19
EV-harvesting Medium
Serum free medium
Separation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Not detected contaminants
Grp78/ Mitofilin
Proteomics
Proteomics database
No
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.05
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
SEC fraction-dependent
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190036 3/3 Homo sapiens Cell culture supernatant Filtration
qEV
Hicks, David 2020 63%

Study summary

Full title
All authors
David A Hicks, Alys C Jones, Nicola J Corbett, Kate Fisher, Stuart M Pickering-Brown, Mark P Ashe, Nigel M Hooper
Journal
Neurochem Res.
Abstract
Healthy brain function is mediated by several complementary signalling pathways, many of which are d (show more...)Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30–100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons. (hide)
EV-METRIC
63% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
OX1-19
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Filtration + qEV
Protein markers
EV: TSG101/ CD9
non-EV: Mitofilin/ Grp78
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
OX1-19
EV-harvesting Medium
Serum free medium
Separation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Not detected contaminants
Mitofilin/ Grp78
Proteomics
Proteomics database
No
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.05
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
SEC fraction-dependent
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190013 1/1 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Johnson, Suzanne M 2020 63%

Study summary

Full title
All authors
Suzanne M Johnson, Antonia Banyard, Christopher Smith, Aleksandr Mironov, Martin G McCabe
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, pro (show more...)Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring. (hide)
EV-METRIC
63% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
UW228-2
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV: CD63/ CD9/ LAMP1
non-EV:
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
UW228-2
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
A-4-38
Pelleting: speed (g)
2000
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
Imagestream
Detected EV-associated proteins
CD63
Detected EV-associated proteins
CD63/ CD9/ LAMP1
Detected EV-associated proteins
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
600-6000
Particle analysis: flow cytometry
Flow cytometer type
Imagestream
Hardware adjustment
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
4500
EV200192 1/2 Exophiala dermatitidis Cell culture supernatant (Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Density gradient
Lavrin, Teja 2020 58%

Study summary

Full title
All authors
Teja Lavrin, Tilen Konte, Rok Kostanjšek, Simona Sitar, Kristina Sepčič, Sonja Prpar Mihevc, Ema Žagar, Vera Župunski, Metka Lenassi, Boris Rogelj, Nina Gunde Cimerman
Journal
Cells
Abstract
The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits d (show more...)The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of E. dermatitidis on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of E. dermatitidis biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of E. dermatitidis with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by E. dermatitidis hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's. (hide)
EV-METRIC
58% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
EXF-10123
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Filtration + Ultrafiltration + Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.09–1.32
Show all info
Study aim
Function
Sample
Species
Exophiala dermatitidis
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
EXF-10123
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Continuous
Lowest density fraction
20
Highest density fraction
60
Total gradient volume, incl. sample (mL)
4.8
Sample volume (mL)
0.4
Orientation
Not specified
Rotor type
Not specified
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.4
Fraction processing
Precipitation
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Protein Concentration Method
BCA
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
Particle analysis: flow cytometry
Hardware adjustment
EM
EM-type
Transmission-EM
Image type
Close-up
Report type
Mean
Report size
90
EV-concentration
Yes
EV200192 2/2 Exophiala dermatitidis Cell culture supernatant (Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Density gradient
Lavrin, Teja 2020 58%

Study summary

Full title
All authors
Teja Lavrin, Tilen Konte, Rok Kostanjšek, Simona Sitar, Kristina Sepčič, Sonja Prpar Mihevc, Ema Žagar, Vera Župunski, Metka Lenassi, Boris Rogelj, Nina Gunde Cimerman
Journal
Cells
Abstract
The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits d (show more...)The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of E. dermatitidis on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of E. dermatitidis biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of E. dermatitidis with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by E. dermatitidis hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's. (hide)
EV-METRIC
58% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
EXF-10123
Sample origin
Melanin inhibitor
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Filtration + Ultrafiltration + Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.09–1.32
Show all info
Study aim
Function
Sample
Species
Exophiala dermatitidis
Sample Type
Cell culture supernatant
Sample Condition
Melanin inhibitor
EV-producing cells
EXF-10123
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Continuous
Lowest density fraction
20
Highest density fraction
60
Total gradient volume, incl. sample (mL)
4.8
Sample volume (mL)
0.4
Orientation
Not specified
Rotor type
Not specified
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.4
Fraction processing
Precipitation
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Protein Concentration Method
BCA
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
Particle analysis: flow cytometry
Hardware adjustment
EM
EM-type
Transmission-EM
Image type
Close-up
Report type
Mean
Report size
75
EV-concentration
Yes
EV200033 1/2 Pseudomonas aeruginosa Cell culture supernatant DG
(d)(U)C
UF
Filtration
Dinh, Nhung Thi Hong 2020 57%

Study summary

Full title
All authors
Nhung Thi Hong Dinh, Jaewook Lee, Jaemin Lee, Sang Soo Kim, Gyeongyun Go, Seoyoon Bae, Ye In Jun, Yae Jin Yoon, Tae-Young Roh, Yong Song Gho
Journal
J Extracell Vesicles
Abstract
Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust cont (show more...)Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust contains extracellular vesicles (EVs), which are associated with pulmonary inflammation. However, it has not been reported whether indoor dust EVs affect the cancer lung metastasis. In this study, we isolated indoor dust EVs and investigated their roles in cancer lung metastasis. Upon intranasal administration, indoor dust EVs enhanced mouse melanoma lung metastasis in a dose-dependent manner in mice. Pre-treatment or co-treatment of indoor dust EVs significantly promoted melanoma lung metastasis, whereas post-treatment of the EVs did not. In addition, the lung lysates from indoor dust EV-treated mice significantly increased tumour cell migration in vitro. We observed that tumour necrosis factor-α played important roles in indoor dust EV-mediated promotion of tumour cell migration in vitro and cancer lung metastasis in vivo. Furthermore, Pseudomonas EVs, the main components of indoor dust EVs, and indoor dust EVs showed comparable effects in promoting tumour cell migration in vitro and cancer lung metastasis in vivo. Taken together, our results suggest that indoor dust EVs, at least partly contributed by Pseudomonas EVs, are potential promoting agents of cancer lung metastasis. (hide)
EV-METRIC
57% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
PAO1
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + UF + Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
N/A
Show all info
Study aim
Function
Sample
Species
Pseudomonas aeruginosa
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PAO1
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
150000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
5.25
Sample volume (mL)
2.5
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
200000
Duration (min)
120
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polysulfone;Other
Protein Concentration Method
Bradford
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
81.9
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190083 1/1 Pectobacterium betavasculorum Cell culture supernatant (d)(U)C
Filtration
Piotrowska, Martyna 2020 57%

Study summary

Full title
All authors
Martyna Piotrowska, Krzesimir Ciura, Michalina Zalewska, Marta Dawid, Bruna Correia, Paulina Sawicka, Bogdan Lewczuk, Joanna Kasprzyk, Laura Sola, Wojciech Piekoszewski, Bartosz Wielgomas, Krzysztof Waleron, Szymon Dziomba
Journal
J Chromatogr A
Abstract
The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were invest (show more...)The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs. (hide)
EV-METRIC
57% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
IFB5271
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
New methodological development/Identification of content (omics approaches)
Sample
Species
Pectobacterium betavasculorum
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
IFB5271
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
195
Pelleting: rotor type
SW 28
Pelleting: speed (g)
85000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Proteomics
Proteomics database
Yes:
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Other particle analysis name(1)
Capillary electrophoresis
Report type
Not Reported
EV-concentration
Yes
EV190052 1/1 Solanum lycopersicum root exudate solution (d)(U)C
Filtration
De Palma, Monica 2020 57%

Study summary

Full title
All authors
Monica De Palma, Alfredo Ambrosone, Antonietta Leone, Pasquale Del Gaudio, Michelina Ruocco, Lilla Turiák, Ramesh Bokka, Immacolata Fiume, Marina Tucci, Gabriella Pocsfalvi
Journal
Plants (Basel)
Abstract
Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Des (show more...)Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Despite their relevant biological implications, the existence and role of plant EVs released into the environment has been unexplored. Herein, we purified round-shaped small vesicles (EVs) by differential ultracentrifugation of a sampling solution containing root exudates of hydroponically grown tomato plants. Biophysical analyses, by means of dynamic light scattering, microfluidic resistive pulse sensing and scanning electron microscopy, showed that the size of root-released EVs range in the nanometric scale (50-100 nm). Shot-gun proteomics of tomato EVs identified 179 unique proteins, several of which are known to be involved in plant-microbe interactions. In addition, the application of root-released EVs induced a significant inhibition of spore germination and of germination tube development of the plant pathogens Fusarium oxysporum, Botrytis cinerea and Alternaria alternata. Interestingly, these EVs contain several proteins involved in plant defense, suggesting that they could be new components of the plant innate immune system. (hide)
EV-METRIC
57% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
root exudate solution
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Solanum lycopersicum
Sample Type
root exudate solution
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
150000
Wash: volume per pellet (ml)
28.5
Wash: time (min)
60
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
150000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Proteomics
Proteomics database
Yes:
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
72 +/- 5 nm
TRPS
Report type
Size range/distribution
Reported size (nm)
51
EV concentration
Yes
EM
EM-type
Scanning electron microscopy
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV190032 1/2 Schistosoma mansoni Schisostomula (larval stage) culture supernatant (d)(U)C Marije E Kuipers 2020 57%

Study summary

Full title
All authors
Marije E Kuipers, Esther N M Nolte-'t Hoen, Alwin J van der Ham, Arifa Ozir-Fazalalikhan, D Linh Nguyen, Clarize M de Korne, Roman I Koning, John J Tomes, Karl F Hoffmann, Hermelijn H Smits, Cornelis H Hokke
Journal
J Extracell Vesicles
Abstract
Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host imm (show more...)Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. (hide)
EV-METRIC
57% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Schisostomula (larval stage) culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Adj. k-factor
213.2 (pelleting) / 213.2 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Mechanism of uptake/transfer, Identification of content (omics approaches)
Sample
Species
Schistosoma mansoni
Sample Type
Schisostomula (larval stage) culture supernatant
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
60
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
213.2
Characterization: Protein analysis
PMID previous EV protein analysis
26443722
Extra characterization
Protein Concentration Method
microBCA
Protein Concentration
6
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-650
EV concentration
Yes
Particle yield
23300000000
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
35-190;30-715
EV190032 2/2 Schistosoma mansoni Schisostomula (larval stage) culture supernatant (d)(U)C Marije E Kuipers 2020 57%

Study summary

Full title
All authors
Marije E Kuipers, Esther N M Nolte-'t Hoen, Alwin J van der Ham, Arifa Ozir-Fazalalikhan, D Linh Nguyen, Clarize M de Korne, Roman I Koning, John J Tomes, Karl F Hoffmann, Hermelijn H Smits, Cornelis H Hokke
Journal
J Extracell Vesicles
Abstract
Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host imm (show more...)Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. (hide)
EV-METRIC
57% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Schisostomula (larval stage) culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Adj. k-factor
213.2 (pelleting) / 83.21 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Mechanism of uptake/transfer, Identification of content (omics approaches)
Sample
Species
Schistosoma mansoni
Sample Type
Schisostomula (larval stage) culture supernatant
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
65
Wash: Rotor Type
TLS-55
Wash: speed (g)
120000
Wash: adjusted k-factor
83.21
Characterization: Protein analysis
PMID previous EV protein analysis
26443722
Extra characterization
Protein Concentration Method
microBCA
Protein Concentration
6
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-650
EV concentration
Yes
Particle yield
23300000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
35-190
EV200182 4/11 Mus musculus Cell culture supernatant (Differential) (ultra)centrifugation
Density cushion
Density gradient
Frühbeis, Carsten 2020 56%

Study summary

Full title
All authors
Carsten Frühbeis, Wen Ping Kuo-Elsner, Christina Müller, Kerstin Barth, Leticia Peris, Stefan Tenzer, Wiebke Möbius, Hauke B Werner, Klaus-Armin Nave, Dominik Fröhlich, Eva-Maria Krämer-Albers
Journal
PLoS Biol
Abstract
Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term in (show more...)Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity. (hide)
EV-METRIC
56% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary oligodendrocytes
Sample origin
PLPko
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Density cushion + Density gradient
Protein markers
EV: SIRT2/ Flotillin1
non-EV: None
Proteomics
yes
EV density (g/ml)
1.07-1.1
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
PLPko
EV-producing cells
primary oligodendrocytes
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.3M
Highest density fraction
1.8M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
60
Pelleting: rotor type
TLS-55
Pelleting: speed (g)
100000
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ SIRT2
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Characterization: Particle analysis
EV200182 7/11 Mus musculus Cell culture supernatant (Differential) (ultra)centrifugation
Density cushion
Density gradient
Frühbeis, Carsten 2020 56%

Study summary

Full title
All authors
Carsten Frühbeis, Wen Ping Kuo-Elsner, Christina Müller, Kerstin Barth, Leticia Peris, Stefan Tenzer, Wiebke Möbius, Hauke B Werner, Klaus-Armin Nave, Dominik Fröhlich, Eva-Maria Krämer-Albers
Journal
PLoS Biol
Abstract
Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term in (show more...)Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity. (hide)
EV-METRIC
56% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary oligodendrocytes
Sample origin
CNPko
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Density cushion + Density gradient
Protein markers
EV: SIRT2/ Flotillin1/ PLP
non-EV: None
Proteomics
yes
EV density (g/ml)
1.07-1,1
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
CNPko
EV-producing cells
primary oligodendrocytes
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.3M
Highest density fraction
1.8M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1ml
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1ml
Fraction processing
Centrifugation
Pelleting: volume per fraction
1ml
Pelleting: duration (min)
60
Pelleting: rotor type
TLS-55
Pelleting: speed (g)
100000
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ PLP/ SIRT2
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Characterization: Particle analysis
EV200080 1/4 Homo sapiens Serum (d)(U)C Gabriella Dobra 2020 56%

Study summary

Full title
All authors
Gabriella Dobra, Matyas Bukva, Zoltan Szabo, Bella Bruszel, Maria Harmati, Edina Gyukity-Sebestyen, Adrienn Jenei, Monika Szucs, Peter Horvath, Tamas Biro, Almos Klekner, Krisztina Buzas
Journal
Int J Mol Sci
Abstract
Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may (show more...)Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen's d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch's test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum. (hide)
EV-METRIC
56% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: Alix/ CD81
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
T-1270
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD81
Proteomics
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
111
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 9.29E+10
EM
EM-type
Atomic force-EM
Image type
Wide-field
EV200080 2/4 Homo sapiens Serum (d)(U)C Gabriella Dobra 2020 56%

Study summary

Full title
All authors
Gabriella Dobra, Matyas Bukva, Zoltan Szabo, Bella Bruszel, Maria Harmati, Edina Gyukity-Sebestyen, Adrienn Jenei, Monika Szucs, Peter Horvath, Tamas Biro, Almos Klekner, Krisztina Buzas
Journal
Int J Mol Sci
Abstract
Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may (show more...)Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen's d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch's test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum. (hide)
EV-METRIC
56% (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 quantit