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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
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
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% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
genetically modified cell line
Focus vesicles
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% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (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% (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
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 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% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
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% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (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 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% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (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 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% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (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 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
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% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (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% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (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 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
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
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 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 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
Glioblastoma multiforme
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
Glioblastoma multiforme
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 database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
108
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 8.19E+10
EM
EM-type
Atomic force-EM
Image type
Wide-field
EV200054 5/7 Mus musculus Brain tissue (d)(U)C
Filtration
Commercial method
Huang, Yiyao 2020 56%

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
56% (58th 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 + Commercial method
Protein markers
EV: Rab27a/ TSG101
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
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Rab27a
Not detected EV-associated proteins
TSG101
Detected contaminants
GM130/ Calnexin
Characterization: Particle analysis
NTA
EV concentration
Yes
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV200036 5/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Progeria patients
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 + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Progeria patients
EV-producing cells
human skin primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 7/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 9/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
iRas
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 + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iRas
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 11/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
iRas+GSTM2
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 + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iRas+GSTM2
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 13/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
iC+GSTM2
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 + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iC+GSTM2
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 15/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
mCherry-CD63
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 + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
mCherry-CD63
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200034 1/1 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation
Density gradient
Filtration
Xiaohui Chen 2020 56%

Study summary

Full title
All authors
Xiaohui Chen, Mei Jia, Lianhua Liu, Xiaopei Qiu, Hong Zhang, Xingle Yu, Wei Gu, Guangchao Qing, Qingmei Li, Xiaolin Hu, Ruixuan Wang, Xianxian Zhao, Liangliang Zhang, Xianfeng Wang, Colm Durkan, Nan Wang, Guixue Wang, Yang Luo
Journal
Small
Abstract
Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for eluci (show more...)Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs' molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs' microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Density gradient + Filtration
Protein markers
EV: TSG101/ CD9/ CD81/ CD63/ actin-beta
non-EV: None
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
New methodological development/Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
A549
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)
120
Pelleting: rotor type
Not reported
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
Not specified
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
2
Fraction processing
Ultracentrifugation
Pelleting: duration (min)
60
Pelleting: rotor type
Not reported
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
TSG101/ CD9/ CD81/ CD63/ actin-beta
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
104
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200024 1/2 Homo sapiens Blood plasma (d)(U)C
Otahal, Alexander 2020 56%

Study summary

Full title
All authors
Alexander Otahal, Karina Kramer, Olga Kuten-Pella, René Weiss, Christoph Stotter, Zsombor Lacza, Viktoria Weber, Stefan Nehrer and Andrea De Luna
Journal
Front. Bioeng. Biotechnol.
Abstract
Autologous blood products gain increasing interest in the field of regenerative medicine as well as (show more...)Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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: Alix/ CD63/ CD9
non-EV: APOA1/ APOB100
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
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)
120
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Alix
Not detected contaminants
APOA1/ APOB100
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
160
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Gallios
Hardware adjustment
Calibration bead size
1
EV200024 2/2 Homo sapiens Serum (d)(U)C Otahal, Alexander 2020 56%

Study summary

Full title
All authors
Alexander Otahal, Karina Kramer, Olga Kuten-Pella, René Weiss, Christoph Stotter, Zsombor Lacza, Viktoria Weber, Stefan Nehrer and Andrea De Luna
Journal
Front. Bioeng. Biotechnol.
Abstract
Autologous blood products gain increasing interest in the field of regenerative medicine as well as (show more...)Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA. (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
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: Alix/ CD63/ CD9
non-EV: APOA1/ APOB100
Proteomics
no
Show all info
Study aim
Function
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 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Alix
Not detected contaminants
APOA1/ APOB100
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
170
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Gallios
Hardware adjustment
Calibration bead size
1
Report type
Modus
Reported size (nm)
170
EV200016 1/2 Bos taurus Cell culture supernatant (d)(U)C
Commercial method
Filtration
Samuel Gebremedhn 2020 56%

Study summary

Full title
All authors
Samuel Gebremedhn, Ahmed Gad, Hoda Samir Aglan, Jozef Laurincik, Radek Prochazka, Dessie Salilew-Wondim, Michael Hoelker, Karl Schellander, Dawit Tesfaye
Journal
Sci Rep
Abstract
Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, wh (show more...)Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Granulosa cells subjected to normal temperature (37OC)
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 + Commercial method + Filtration
Protein markers
EV: CD63
non-EV: Cytochrome C
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
Cell culture supernatant
Sample Condition
Granulosa cells subjected to normal temperature (37OC)
EV-producing cells
Ovarian granulosa cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
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)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
5
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63
Not detected contaminants
Cytochrome C
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
131
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 5.98E+08
EM
EM-type
Transmission-EM
Image type
Close-up
EV200016 2/2 Bos taurus Cell culture supernatant (d)(U)C
Commercial method
Filtration
Samuel Gebremedhn 2020 56%

Study summary

Full title
All authors
Samuel Gebremedhn, Ahmed Gad, Hoda Samir Aglan, Jozef Laurincik, Radek Prochazka, Dessie Salilew-Wondim, Michael Hoelker, Karl Schellander, Dawit Tesfaye
Journal
Sci Rep
Abstract
Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, wh (show more...)Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Granulosa cells subjected to higher temperature (42OC)
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 + Commercial method + Filtration
Protein markers
EV: CD63
non-EV: Cytochrome
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
Cell culture supernatant
Sample Condition
Granulosa cells subjected to higher temperature (42OC)
EV-producing cells
Ovarian granulosa cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
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)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
5
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63
Not detected contaminants
Cytochrome
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
129
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 7.23E+08
EM
EM-type
Transmission-EM
Image type
Close-up
EV200012 2/2 Rattus norvegicus Cell culture supernatant Ultrafiltration Doreen Matthies 2020 56%

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
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Ultrafiltration
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
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
Cryo-EM
Image type
Close-up
Report size (nm)
147.82+/-95.72
EV concentration
Yes
EV200000 1/4 Homo sapiens pleural effusion (d)(U)C Ping, Luo 2020 56%

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
56% (57th 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
tuberculosis
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
tuberculosis
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
CD63/ TSG101/ CD81
Not detected EV-associated proteins
CD9
Not detected contaminants
calnexin/ GM130
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
136.1
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 182567942
EV200000 2/4 Homo sapiens pleural effusion (d)(U)C Ping, Luo 2020 56%

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
56% (57th 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
tuberculosis
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
tuberculosis
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)
224.2
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 244682871
EV190089 1/1 Homo sapiens Cell culture supernatant (d)(U)C Guowen Hu 2020 56%

Study summary

Full title
All authors
Guowen Hu, Yuguo Xia, Juntao Zhang, Yu Chen, Ji Yuan, Xin Niu, Bizeng Zhao, Qing Li, Yang Wang, Zhifeng Deng
Journal
Advanced Science
Abstract
Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism (show more...)Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism is unclear and there is still lack of effective medications. In this study, the VD rats exhibit a progressive cognitive impairment, as well as a time‐related increasing in hippocampal neural stem cells (H‐NSCs) senescence, lost and neurogenesis decline. Then, embryonic stem cell‐derived small extracellular vesicles (ESC‐sEVs) are intravenously injected into VD rats. ESC‐sEVs treatment significantly alleviates H‐NSCs senescence, recovers compromised proliferation and neuron differentiation capacity, and reverses cognitive impairment. By microarray analysis and RT‐qPCR it is identified that several miRNAs including miR‐17‐5p, miR‐18a‐5p, miR‐21‐5p, miR‐29a‐3p, and let‐7a‐5p, that can inhibit mTORC1 activation, exist in ESC‐sEVs. ESC‐sEVs rejuvenate H‐NSCs senescence partly by transferring these miRNAs to inhibit mTORC1 activation, promote transcription factor EB (TFEB) nuclear translocation and lysosome resumption. Taken together, these data indicate that H‐NSCs senescence cause cell depletion, neurogenesis reduction, and cognitive impairment in VD. ESC‐sEVs treatment ameliorates H‐NSCs senescence by inhibiting mTORC1 activation, and promoting TFEB nuclear translocation and lysosome resumption, thereby reversing senescence‐related neurogenesis dysfunction and cognitive impairment in VD. The application of ESC‐sEVs may be a novel cell‐free therapeutic tool for patients with VD, as well as other aging‐related diseases. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Embryonic stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
114
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38.5
Wash: time (min)
114
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected contaminants
GM130
EV190069 3/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Mariscal, Javier 2020 56%

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
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
DIAPH3 Knock down
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: CD9/ HSPA5
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
DIAPH3 Knock down
EV-producing cells
DU145
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 660 nm
Western Blot
Detected EV-associated proteins
HSPA5
Not detected EV-associated proteins
CD9
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
1650
EV concentration
Yes
EV190069 4/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Mariscal, Javier 2020 56%

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
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
DIAPH3 Knock down
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: CD9/ HSPA5
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
DIAPH3 Knock down
EV-producing cells
DU145
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 660 nm
Western Blot
Detected EV-associated proteins
CD9
Not detected EV-associated proteins
HSPA5
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
116
EV concentration
Yes
EV190066 1/2 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Gori, Alessandro 2020 56%

Study summary

Full title
All authors
Alessandro Gori, Alessandro Romanato, Bergamaschi Greta, Alessandro Strada, Paola Gagni, Roberto Frigerio, Dario Brambilla, Riccardo Vago, Silvia Galbiati, Silvia Picciolini, Marzia Bedoni, George G. Daaboul, Marcella Chiari, and Marina Creticha
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extrac (show more...)Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis. (hide)
EV-METRIC
56% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + qEV
Protein markers
EV: TSG101/ Alix/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HEK
EV-harvesting Medium
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Surespin 630 (17 ml)
Pelleting: speed (g)
28400
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
203
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190066 2/2 Homo sapiens Serum (d)(U)C
qEV
Gori, Alessandro 2020 56%

Study summary

Full title
All authors
Alessandro Gori, Alessandro Romanato, Bergamaschi Greta, Alessandro Strada, Paola Gagni, Roberto Frigerio, Dario Brambilla, Riccardo Vago, Silvia Galbiati, Silvia Picciolini, Marzia Bedoni, George G. Daaboul, Marcella Chiari, and Marina Creticha
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extrac (show more...)Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis. (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
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 + qEV
Protein markers
EV: TSG101/ Alix/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
41900
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected EV-associated proteins
TSG101/ CD63/ CD9/ Alix
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
208
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190064 2/10 Homo sapiens Urine (d)(U)C
Filtration
Dhondt B 2020 56%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
56% (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
Urine
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: Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
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
SW 32.1 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
16
Wash: time (min)
70
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ CD9
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
226.2
EV concentration
Yes
EV190060 1/4 Homo sapiens Blood plasma (d)(U)C Mari Palviainen 2020 56%

Study summary

Full title
All authors
Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
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)
90
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
20
Wash: time (min)
90
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD41/ TSG101
Flow cytometry aspecific beads
Detected EV-associated proteins
CD61/ CD235a/ phosphatidylserine
Proteomics database
Yes:
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
107-145
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Apogee A50
Hardware adjustment
calibration done with apogee Mix beads 80-1300 nm
Calibration bead size
80
EM
EM-type
Transmission-EM
Image type
Close-up
EV190060 2/4 Homo sapiens Blood plasma (d)(U)C Mari Palviainen 2020 56%

Study summary

Full title
All authors
Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
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)
90
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
20
Wash: time (min)
90
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD41/ TSG101
Flow cytometry aspecific beads
Detected EV-associated proteins
CD61/ CD235a/ phosphatidylserine
Proteomics database
Yes:
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
117-162
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
apogee A50
Hardware adjustment
calibration done with apogee Mix beads 80-1300 nm
Calibration bead size
80
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up
EV190060 3/4 Homo sapiens Blood plasma (d)(U)C Mari Palviainen 2020 56%

Study summary

Full title
All authors
Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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