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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Vesicle type
Experiment number
  • Experiments differ in Vesicle type
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  • Experiments differ in Sample type, Isolation method
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Experiment number
  • Experiments differ in Sample type, Isolation method
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV170030 1/1 Mus musculus Cell culture supernatant DG
dUC
Driedonks, Tom A. P. 2018 100%

Study summary

Full title
All authors
Driedonks TAP, van der Grein SG, Ariyurek Y, Buermans HPJ, Jekel H, Chow FWN, Wauben MHM, Buck AH, 't Hoen PAC, Nolte-'t Hoen ENM
Journal
Cell Mol Life Sci
Abstract
The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of inte (show more...)The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: CD9/ CD63/ MHC2/ Galectin-3
non-EV: beta-actin
Proteomics
no
Show all info
Study aim
Biomarker, Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary bone marrow dendritic cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
90
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Sample volume (mL)
0.15
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900-1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Pelleting: adjusted k-factor
144.0
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, MHC2, Galectin-3
Not detected contaminants
beta-actin
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100 - 200
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine
Calibration bead size
0.1,0.2
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170013 1/1 Mus musculus Cell culture supernatant DG
dUC
Degosserie, Jonathan 2018 100%

Study summary

Full title
All authors
Degosserie J, Heymans C, Spourquet C, Halbout M, D'Auria L, Van Der Smissen P, Vertommen D, Courtoy PJ, Tyteca D, Pierreux CE.
Journal
J Extracell Vesicles
Abstract
Organogenesis is a complex and dynamic process requiring reciprocal communication between different (show more...)Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-α1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-α1. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
84.53 (pelleting) / 84.53 (washing)
Protein markers
EV: CD9/ CD63/ Flotillin-1
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
endothelial progenitor cells
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Pelleting: adjusted k-factor
84.53
Wash: time (min)
90
Wash: Rotor Type
Type 80 Ti
Wash: speed (g)
150000
Wash: adjusted k-factor
84.53
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
30%
Sample volume (mL)
1.3
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 55 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0.625
Fraction processing
Centrifugation
Pelleting: volume per fraction
8
Pelleting: duration (min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Pelleting: adjusted k-factor
84.53
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-150
EV concentration
Yes
Particle yield
3.60E+08 particles/million cells
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV170020 1/2 Trypanosoma cruzi Cell culture supernatant DG
dUC
Filtration
Caeiro, Lucas 2018 77%

Study summary

Full title
All authors
Caeiro LD, Alba-Soto CD, Rizzi M, Solana ME, Rodriguez G, Chidichimo AM, Rodriguez ME, Sánchez DO, Levy GV, Tekiel V.
Journal
PLoS Negl Trop Dis
Abstract
TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage (show more...)TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes. (hide)
EV-METRIC
77% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: HSP70/ TcTASV-C
non-EV: TcSR-62
Proteomics
yes
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Trypanosoma cruzi
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA
EV-harvesting Medium
Serum free medium
Cell viability
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
2
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Concentration
0.1
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70,TcTASV-C
Not detected contaminants
TcSR-62
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-150
EV170020 2/2 Trypanosoma cruzi Cell culture supernatant DG
dUC
Filtration
Caeiro, Lucas 2018 77%

Study summary

Full title
All authors
Caeiro LD, Alba-Soto CD, Rizzi M, Solana ME, Rodriguez G, Chidichimo AM, Rodriguez ME, Sánchez DO, Levy GV, Tekiel V.
Journal
PLoS Negl Trop Dis
Abstract
TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage (show more...)TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes. (hide)
EV-METRIC
77% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: HSP70/ TcTASV-C
non-EV: TcSR-62
Proteomics
yes
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Trypanosoma cruzi
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA
EV-harvesting Medium
Serum free medium
Cell viability
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
1080
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
1080
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
2
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Concentration
0.15
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70,TcTASV-C
Not detected contaminants
TcSR-62
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-150
EV180022 1/4 Homo sapiens Cell culture supernatant dUC Kaur S 2018 67%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
67% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
pluripotent stem cells
EV-harvesting Medium
Serum free medium
Cell viability
80
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
101-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180022 4/4 Homo sapiens Cell culture supernatant Commercial method Kaur S 2018 67%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
67% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
pluripotent stem cells
EV-harvesting Medium
Serum free medium
Cell viability
80
Isolation Method
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)
Protein Concentration Method
Not determined
Fluorescent NTA
EV180022 3/4 Homo sapiens Cell culture supernatant dUC
Filtration
Kaur S 2018 66%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
66% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: CD63/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
adipose tissue-derived mesenchymal stem cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
80
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, TSG101, CD90
Not detected contaminants
Calnexin
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
101-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180017 1/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 63%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
63% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63/ Flotillin-2/ TSG101/ MT1-MMP
non-EV: PMP70/ CytochromeC/ Calnexin/ Golgin245
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-2, TSG101, MT1-MMP
Not detected contaminants
PMP70, CytochromeC, Calnexin, Golgin245
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131
EV concentration
Yes
Particle yield
4.95E+09 particles/million cells
EV180017 2/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 62%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
62% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing fascin nanobody
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
142
EV concentration
Yes
Particle yield
2.25E+10 particles/million cells
EV180017 5/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 62%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
62% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing cortactin nanobody (NTA domain)
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133
EV concentration
Yes
Particle yield
1.16E+09 particles/million cells
EV170025 1/1 Homo sapiens Urine Commercial method
Ultrafiltration
Oeyen E 2018 62%

Study summary

Full title
All authors
Oeyen E, Van Mol K, Baggerman G, Willems H, Boonen K, Rolfo C, Pauwels P, Jacobs A, Schildermans K, Cho WC, Mertens I.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolati (show more...)Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs. (hide)
EV-METRIC
62% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + Ultrafiltration
Protein markers
EV: Flotillin-1
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Isolation Method
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Concentration
0.81
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin-1
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
108
EV concentration
Yes
Particle yield
1.80E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
40-100
Other particle analysis name(1)
Asymmetrical flow field-flow fractionation
Report type
Size range/distribution
Report size
40-160
EV-concentration
No
EV180044 1/1 Homo sapiens Cell culture supernatant dUC
Filtration
Biscans A 2018 55%

Study summary

Full title
All authors
Biscans A, Haraszti RA, Echeverria D, Miller R, Didiot MC, Nikan M, Roux L, Aronin N, Khvorova A
Journal
J Cell Sci
Abstract
Small extracellular vesicles (sEVs) show promise as natural nano-devices for delivery of therapeutic (show more...)Small extracellular vesicles (sEVs) show promise as natural nano-devices for delivery of therapeutic RNA, but efficient loading of therapeutic RNA remains a challenge. We have recently shown that the attachment of cholesterol to small interfering RNAs (siRNAs) enables efficient and productive loading into sEVs. Here, we systematically explore the ability of lipid conjugates-fatty acids, sterols, and vitamins-to load siRNAs into sEVs and support gene silencing in primary neurons. Hydrophobicity of the conjugated siRNAs defined loading efficiency and the silencing activity of siRNA-sEVs complexes. Vitamin-E-conjugated siRNA supported the best loading into sEVs and productive RNA delivery to neurons. (hide)
EV-METRIC
55% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
209.7 (pelleting) / 65.69 (washing)
Protein markers
EV: CD63/ CD81
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Mechanism of uptake/transfer, 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
PCS-500-010
EV-harvesting Medium
Serum free medium
Cell viability
100
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
209.7
Wash: time (min)
90
Wash: Rotor Type
TLA-110
Wash: speed (g)
100000
Wash: adjusted k-factor
65.69
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81
Not detected contaminants
Calnexin
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
140
EV concentration
Yes
Particle yield
3.20E+08 particles/million cells
EM
EM-type
Transmission-EM
Image type
Wide-field
Extra information
Rotor types and NTA data added post-publication.
EV180042 1/1 Mus musculus Cell culture supernatant dUC Ikebuchi, Yuki 2018 55%

Study summary

Full title
All authors
Ikebuchi Y, Aoki S, Honma M, Hayashi M, Sugamori Y, Khan M, Kariya Y, Kato G, Tabata Y, Penninger JM, Udagawa N, Aoki K, Suzuki H.
Journal
Nature
Abstract
Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of oste (show more...)Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis. (hide)
EV-METRIC
55% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
209.7 (pelleting) / 209.7 (washing)
Protein markers
EV: CD9/ CD63/ CD81/ RANK
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
RANKL-stimulated
EV-producing cells
RAW264.7,mouse primary osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
50
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
209.7
Wash: time (min)
60
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
209.7
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
2.5-3.5
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, CD81
Not detected contaminants
Calnexin
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
Yes
Fluorescent NTA
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up
Extra information
Antibody details for Western blot: rabbit anti-CD9 (Abcam, CatNo. ab92726, clone EPR2949, LotNo. GR260186-10, 1:1000), rat anti-CD63 (MBL, CatNo. D263-3, clone R5G2, LotNo. 014, 1:500), hamster anti-CD81 (Bio-Rad, CatNo. MCA1846GA, clone Eat2, LotNo. 0515, 1:500), rabbit anti-Calnexin (Abcam, CatNo. ab22595, polyclonal, LotNo. GR86850-1, 1:500)
EV180013 1/1 Homo sapiens Blood plasma dUC Malin, Steven 2018 55%

Study summary

Full title
All authors
Eichner NZM, Gilbertson NM, Gaitan JM, Heiston EM, Musante L, LaSalvia S, Weltman A, Erdbrügger U, Malin SK
Journal
Physiol Rep
Abstract
Low cardiorespiratory fitness (CRF) is associated with cardiovascular disease (CVD) independent of o (show more...)Low cardiorespiratory fitness (CRF) is associated with cardiovascular disease (CVD) independent of obesity. Extracellular vesicles (EVs) are a novel target of CVD, however, it remains unknown if obese individuals with very poor fitness (VPF) have elevated EVs versus people with poor fitness (PF). Thus, we tested whether VPF was associated with greater EV subtypes in obese adults. Subjects with VPF (n = 13, VO2 peak: 15.4 ± 0.6 mL/kg/min, BMI: 34.1 ± 1.7 kg/m2 ) and PF (n = 13, VO2 peak: 25.9 ± 3.0 mL/kg/min, BMI: 32.1 ± 1.2 kg/m2 ) were compared in this cross-sectional study. After an overnight fast, AnnexinV (AV) +/- platelet (CD31+ /CD41+ ), leukocyte (CD45+ /CD41- ), and endothelial EVs (CD105+ , CD31+ /CD41- ) were analyzed from fresh platelet poor plasma via imaging flow cytometry. Body fat, blood pressure (BP), and glucose tolerance (OGTT) were also tested. Body weight, BP, and circulating glucose were similar between groups, although VPF subjects were older than PF (64.0 ± 2.1 vs. 49.8 ± 4.2 year; P < 0.05). People with VPF, compared with PF, had higher total AV- EVs (P = 0.04), AV- platelet EVs (CD31+ /CD41+ ; P = 0.006), and AV- endothelial EVs (CD31+ /CD41- ; P = 0.005) independent of age and body fat. Higher AV- platelet and endothelial EVs were associated with lower VO2 peak (r = -0.56, P = 0.006 and r = -0.55, P = 0.005, respectively). Endothelial-derived AV- /CD31+ /CD41- EVs were also related to pulse pressure (r = 0.45, P = 0.03), whereas AV- /CD105 was linked to postprandial glucose (r = 0.41, P = 0.04). VPF is associated with higher AnnexinV- total, endothelial, and platelet EVs in obese adults, suggesting that subtle differences in fitness may reduce type 2 diabetes and CVD risk through an EV-related mechanism. (hide)
EV-METRIC
55% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
113.7 (pelleting) / 379.2 (washing)
Protein markers
EV: CD9/ TSG101/ CD31/ CD41/ CD45/ CD105
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Prediabetes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
15
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
50000
Pelleting: adjusted k-factor
113.7
Wash: time (min)
10
Wash: Rotor Type
FA-45-24-11
Wash: speed (g)
15000
Wash: adjusted k-factor
379.2
Characterization: Protein analysis
PMID previous EV protein analysis
Western blot
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, TSG101
Flow cytometry
Type of Flow cytometry
ImageStream MKII Imaging Flow Cytometer
Hardware adjustments
Hardware adaptation is not needed for ImageStream. Controls included single stained EV samples, buffer only controls (collected for 2 min after filtering with a 0.1 µm filter), buffer plus antibodies
Calibration bead size
0.22
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
168
TRPS
Report type
Mean
Reported size (nm)
182
EM
EM-type
Cryo-EM
Image type
Wide-field
EV180065 1/1 Homo sapiens Cell culture supernatant Ultrafiltration
Filtration
(Differential) (ultra)centrifugation
Density gradient
Deschamps T 2018 50%

Study summary

Full title
All authors
Deschamps T, Kalamvoki M
Journal
J Virol
Abstract
Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to u (show more...)Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to uninfected cells viral factors and host components, such as the stimulator of interferon genes (STING), which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1-infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING, along with the EV markers CD63 and CD9, was found in light-density fractions, while HSV components accumulated in heavy-density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected cells, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells, and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1-infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased the HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1-infected cells negatively impact the infection and could control the dissemination of the virus.IMPORTANCE Extracellular vesicles (EVs) are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced, so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1-infected cells carry innate immune components such as STING and other host and viral factors; they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1-infected cells could control HSV-1 dissemination promoting its persistence in the host. (hide)
EV-METRIC
50% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Ultrafiltration + Filtration + (Differential) (ultra)centrifugation + Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Mechanism of uptake/transfer/Biogenesis/cargo sorting/New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HSV-1 (herpes simplex 1 virus)-infected
EV-producing cells
HEL
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
11
Lowest density fraction
18%
Highest density fraction
6%
Total gradient volume, incl. sample (mL)
12
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
250000
Duration (min)
120
Fraction volume (mL)
0.5
Fraction processing
Dialysis and ultrafiltration
Ultra filtration
Cut-off size (kDa)
30
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin2/ CD9/ TSG101/ CD63/ STING
Fluorescent NTA
Relevant measurements variables specified?
NA
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
180
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180017 3/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 50%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
50% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing cortactin nanobody (SH3 domain)
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133
EV concentration
Yes
Particle yield
9.79E+09 particles/million cells
EV180017 4/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 50%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
50% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing gelsolin nanobody
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
128
EV concentration
Yes
Particle yield
1.97E+09 particles/million cells
EV170037 1/3 Homo sapiens Serum dUC Klump, Jennifer 2018 28%

Study summary

Full title
All authors
Klump J, Phillipp U, Follo M, Eremin A, Lehmann H, Nestel S, von Bubnoff N, Nazarenko I
Journal
Nanomedicine
Abstract
Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood bi (show more...)Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. (hide)
EV-METRIC
28% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
213.2 (pelleting) / 213.2 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
120
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
213.2
Protein Concentration Method
microBCA
Protein Concentration
10-50 dependent whether healthy donors or cancer patients were analyzed
Fluorescent NTA
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
different populations of vesicles were detected by DLS and NTA;120-500 nm; and over 1000 nm
NTA
Report type
Size range/distribution
Reported size (nm)
90-300
EV concentration
Yes
Particle yield
3.00E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
90-120
Extra information
Publication aimed to determine the content of mutated DNA oncogenes copy inside of the vesicles (post- DNase treatments) and in the free-circulating fractions. For that DNA was isolated from different EV and fc fractions and DNA was analyzed using ddPCR. Conclusion was that the mutated BRAF and c-KIT copies are preferably located in the free-circulating fractions and not in EVs.
EV170037 2/3 Homo sapiens Serum dUC Klump, Jennifer 2018 28%

Study summary

Full title
All authors
Klump J, Phillipp U, Follo M, Eremin A, Lehmann H, Nestel S, von Bubnoff N, Nazarenko I
Journal
Nanomedicine
Abstract
Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood bi (show more...)Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. (hide)
EV-METRIC
28% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
213.2 (pelleting) / 213.2 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Melanoma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
120
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
213.2
Protein Concentration Method
microBCA
Protein Concentration
10-50 dependent whether healthy donors or cancer patients were analyzed
Fluorescent NTA
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
different populations of vesicles were detected by DLS and NTA;120-500 nm; and over 1000 nm
NTA
Report type
Size range/distribution
Reported size (nm)
90-300
EV concentration
Yes
Particle yield
3.00E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
90-120
Extra information
Publication aimed to determine the content of mutated DNA oncogenes copy inside of the vesicles (post- DNase treatments) and in the free-circulating fractions. For that DNA was isolated from different EV and fc fractions and DNA was analyzed using ddPCR. Conclusion was that the mutated BRAF and c-KIT copies are preferably located in the free-circulating fractions and not in EVs.
EV170037 3/3 Homo sapiens Serum dUC Klump, Jennifer 2018 28%

Study summary

Full title
All authors
Klump J, Phillipp U, Follo M, Eremin A, Lehmann H, Nestel S, von Bubnoff N, Nazarenko I
Journal
Nanomedicine
Abstract
Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood bi (show more...)Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. (hide)
EV-METRIC
28% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
213.2 (pelleting) / 213.2 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Mastocytosis
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
120
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
213.2
Protein Concentration Method
microBCA
Protein Concentration
10-50 dependent whether healthy donors or cancer patients were analyzed
Fluorescent NTA
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
different populations of vesicles were detected by DLS and NTA;120-500 nm; and over 1000 nm
NTA
Report type
Size range/distribution
Reported size (nm)
90-300
EV concentration
Yes
Particle yield
3.00E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
90-120
Extra information
Publication aimed to determine the content of mutated DNA oncogenes copy inside of the vesicles (post- DNase treatments) and in the free-circulating fractions. For that DNA was isolated from different EV and fc fractions and DNA was analyzed using ddPCR. Conclusion was that the mutated BRAF and c-KIT copies are preferably located in the free-circulating fractions and not in EVs.
EV180022 2/4 Homo sapiens Cell culture supernatant Commercial method Kaur S 2018 0%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
adipose tissue-derived mesenchymal stem cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
80
Isolation Method
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)
Protein Concentration Method
Not determined
Fluorescent NTA
EV170015 1/2 Homo sapiens Cell culture supernatant dUC
Filtration
Polymer-based precipitation
Tabak, Saray 2018 0%

Study summary

Full title
All authors
Tabak S, Schreiber-Avissar S, Beit-Yannai E.
Journal
J Cell Mol Med
Abstract
The role of extracellular vesicles (EVs) as signal mediators has been described in many biological f (show more...)The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non-pigmented ciliary epithelium (NPCE)-derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose-response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE-derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (β-Catenin, Axin2 and LEF1) and protein (β-Catenin, GSK-3β) expression using real-time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of β-Catenin, GSK-3β, as opposed to exposure to high exosomal concentrations. Pro-MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE- or RPE-derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration-dependent at their target site. Specifically in the present study, we described a general dose-response at the gene and MMPs activity and a different dose-response regarding key canonical Wnt proteins expression. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + Polymer-based precipitation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
NPCE cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Other
Name other isolation method
50% PEG-8000, 0.5M NaCl, mixed with the conditioned medium 1:5 v/v respectively and incubated overnight at 4°C. The mixtures were centrifuged at 1500g for 30 minutes to pellet the EVs.
Protein Concentration Method
Bradford
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
98±10
EV concentration
Yes
Extra information
The precipated pellet containing the EVs was re-suspended in PBS and pelleted by ultra-centrifugation of 100,000g for 70 minutes at 4°C. The final EVs pelleted were suspended in 1mL PBS and were stored at -80˚C till use.
EV170015 2/2 Homo sapiens Cell culture supernatant dUC
Filtration
Polymer-based precipitation
Tabak, Saray 2018 0%

Study summary

Full title
All authors
Tabak S, Schreiber-Avissar S, Beit-Yannai E.
Journal
J Cell Mol Med
Abstract
The role of extracellular vesicles (EVs) as signal mediators has been described in many biological f (show more...)The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non-pigmented ciliary epithelium (NPCE)-derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose-response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE-derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (β-Catenin, Axin2 and LEF1) and protein (β-Catenin, GSK-3β) expression using real-time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of β-Catenin, GSK-3β, as opposed to exposure to high exosomal concentrations. Pro-MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE- or RPE-derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration-dependent at their target site. Specifically in the present study, we described a general dose-response at the gene and MMPs activity and a different dose-response regarding key canonical Wnt proteins expression. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + Polymer-based precipitation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
RPE cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Other
Name other isolation method
50% PEG-8000, 0.5M NaCl, mixed with the conditioned medium 1:5 v/v respectively and incubated overnight at 4°C. The mixtures were centrifuged at 1500g for 30 minutes to pellet the EVs.
Protein Concentration Method
Bradford
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
128±11
EV concentration
Yes
Extra information
The precipated pellet containing the EVs was re-suspended in PBS and pelleted by ultra-centrifugation of 100,000g for 70 minutes at 4°C. The final EVs pelleted were suspended in 1mL PBS and were stored at -80˚C till use.
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