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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Experiment number
  • Experiments differ in Sample type
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV210134 2/4 Haemophilus parasuis Cell culture supernatant DG
(d)(U)C
Filtration
McCaig, William D 2016 33%

Study summary

Full title
All authors
William D McCaig, Crystal L Loving, Holly R Hughes, Susan L Brockmeier
Journal
PLoS One
Abstract
Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swin (show more...)Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Haemophilus parasuis Nagasaki
Sample origin
Liquid culture
Focus vesicles
Outer membrane vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: Nagasaki bacterin
non-EV: None
Proteomics
yes
EV density (g/ml)
Not specified
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Haemophilus parasuis
Sample Type
Cell culture supernatant
Sample Condition
Liquid culture
EV-producing cells
Haemophilus parasuis Nagasaki
EV-harvesting Medium
Serum-containing medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
30
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
110000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
7
Lowest density fraction
15%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.5
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
30
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Nagasaki bacterin
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
50-200
EV210134 3/4 Haemophilus parasuis Cell culture supernatant DG
(d)(U)C
Filtration
McCaig, William D 2016 33%

Study summary

Full title
All authors
William D McCaig, Crystal L Loving, Holly R Hughes, Susan L Brockmeier
Journal
PLoS One
Abstract
Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swin (show more...)Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Haemophilus parasuis D74
Sample origin
Plate
Focus vesicles
Outer membrane vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: Nagasaki bacterin
non-EV: None
Proteomics
yes
EV density (g/ml)
Not specified
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Haemophilus parasuis
Sample Type
Cell culture supernatant
Sample Condition
Plate
EV-producing cells
Haemophilus parasuis D74
EV-harvesting Medium
Serum-containing medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
30
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
110000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
7
Lowest density fraction
15%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.5
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
30
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Nagasaki bacterin
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
50-200
EV210134 4/4 Haemophilus parasuis Cell culture supernatant DG
(d)(U)C
Filtration
McCaig, William D 2016 33%

Study summary

Full title
All authors
William D McCaig, Crystal L Loving, Holly R Hughes, Susan L Brockmeier
Journal
PLoS One
Abstract
Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swin (show more...)Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Haemophilus parasuis Nagasaki
Sample origin
Plate
Focus vesicles
Outer membrane vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: Nagasaki bacterin
non-EV: None
Proteomics
yes
EV density (g/ml)
Not specified
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Haemophilus parasuis
Sample Type
Cell culture supernatant
Sample Condition
Plate
EV-producing cells
Haemophilus parasuis Nagasaki
EV-harvesting Medium
Serum-containing medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
30
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
110000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
7
Lowest density fraction
15%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.5
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
30
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Nagasaki bacterin
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
50-200
EV210106 5/5 Homo sapiens Urine (d)(U)C
Filtration
Andreu, Zoraida 2016 33%

Study summary

Full title
All authors
Zoraida Andreu, Renan Otta Oshiro, Alberto Redruello, Soraya López-Martín, Cristina Gutiérrez-Vázquez, Esperanza Morato, Ana Isabel Marina, Carlos Olivier Gómez, María Yáñez-Mó
Journal
Eur J Pharm Sci.
Abstract
Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Cu (show more...)Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. (hide)
EV-METRIC
33% (65th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Bladder cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: Filamin-A/ ApoE/ ApoB/ CD9/ ERM
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Bladder cancer
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
JS-24.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
33
Wash: time (min)
60
Wash: Rotor Type
JS-24.38
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
ERM/ ApoB/ CD9
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Detected EV-associated proteins
Filamin-A/ ApoE/ ApoB
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
130
EM
EM-type
Transmission-EM
Image type
Wide-field
EV210103 4/4 Homo sapiens Urine (d)(U)C Lin, Shih-Yi 2016 33%

Study summary

Full title
All authors
Shih-Yi Lin, Chao-Hsiang Chang, His-Chin Wu, Ching-Chan Lin, Kai-Po Chang, Chi-Rei Yang, Chi-Ping Huang, Wu-Huei Hsu, Chiz-Tzung Chang, Chao-Jung Chen
Journal
Sci Rep
Abstract
MALDI-TOF spectrometry has not been used for urinary exosome analysis. We used it for determining UC (show more...)MALDI-TOF spectrometry has not been used for urinary exosome analysis. We used it for determining UC biomarkers. From 2012 to 2015, we enrolled 129 consecutive patients with UC and 62 participants without UC. Exosomes from their urine were isolated, and analyzed through MALDI-TOF spectrometry. Immunohistochemical (IHC) analysis of another 122 UC and 26 non-UC tissues was conducted to verify the discovered biomarkers. Two peaks at m/z 5593 (fragmented peptide of alpha-1-antitrypsin; sensitivity, 50.4%; specificity, 96.9%) and m/z 5947 (fragmented peptide of histone H2B1K sensitivity, 62.0%; specificity, 92.3%) were identified as UC diagnosis exosome biomarkers. UC patients with detectable histone H2B1K showed 2.29- and 3.11-fold increased risks of recurrence and progression, respectively, compared with those with nondetectable histone H2B1K. Verification results of IHC staining revealed significantly higher expression of alpha 1-antitrypsin (p = 0.038) and H2B1K (p = 0.005) in UC tissues than in normal tissues. The expression of alpha 1-antitrypsin and H2B1K in UC tissues was significantly correlated with UC grades (p < 0.05). Urinary exosome proteins alpha 1-antitrypsin and histone H2B1K, which are identified through MALDI-TOF analysis, could facilitate rapid diagnosis and prognosis of UC. (hide)
EV-METRIC
33% (65th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Urothelial carcinoma
Focus vesicles
microparticle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alix/ TSG101/ ?-actin
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Urothelial carcinoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
200000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Alix/ ?-actin/ TSG101
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210077 1/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
RD
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
RD
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g; 0.22 µm filtration
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
GAPDH/ HSC70/ TSG101
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-100
EV210077 2/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
RH36
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
RH36
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g; 0.22 µm filtration
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ TSG101
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-100
EV210077 3/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
RH41
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: HSC70/ GAPDH/ TSG101/ Pax3-FOXO1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
RH41
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g; 0.22 µm filtration
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ TSG101
Not detected EV-associated proteins
Pax3-FOXO1
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-100
EV210077 4/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
JR1
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
JR1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g; 0.22 µm filtration
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ TSG101
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-120
EV210077 5/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
RH30
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ GAPDH/ HSC70/ Pax3-FOXO1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
RH30
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g; 0.22 µm filtration
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ TSG101
Not detected EV-associated proteins
Pax3-FOXO1
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-120
EV200178 1/4 Homo sapiens Blood plasma (d)(U)C
DC
Filtration
Pillay, Preenan 2016 33%

Study summary

Full title
All authors
Preenan Pillay, Niren Maharaj, Jagidesa Moodley, Irene Mackraj
Journal
Placenta
Abstract
Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biolog (show more...)Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biological cells under normal and pathological conditions. Although there have been reports of circulating exosomes in normal pregnancy, the relevance of placental-derived exosomes in normal and abnormal pregnancies still needs to be elucidated. The aim of this study was to quantify total and placental-derived exosomes in maternal plasma from normal (N), early onset- and late onset-preeclampsia (PE). Method: Plasma samples were obtained from pregnant women in the third trimester, for the isolation of exosomes by differential ultracentrifugation. Total exosomes were quantified using nanoparticle tracking analysis and immuno-reactive exosomal CD63 quantification. Placental-derived exosomes were quantified using placental alkaline phosphatase (PLAP) as a specific marker. The contribution of placental-derived exosomes to total exosomes in maternal plasma was determined by the ratio of PLAP+ exosomes to CD63+ exosomes. Results: The concentration of total exosomes significantly increased in early onset-PE and late onset-PE compared to N (≤33 weeks) and N (≥34 weeks). The relative concentration of placental-derived exosomes significantly increased in early onset-PE but decreased in late onset-PE compared to N. The ratio of PLAP+ exosomes to total number of exosomes significantly decreased in early onset-PE and late onset-PE. A positive correlation between total and placental-derived exosomes were obtained in N (≤33 weeks: Pearson's r = 0.60, ≥34 weeks: Pearson's r = 0.67) and early onset-PE (Pearson's r = 0.51, p < 0.05) with the inverse in late onset-PE (Pearson's r = -0.62, p < 0.01). Conclusion: The differences in the contribution of placental-derived exosomes to total exosomes in maternal circulation suggests a possible pathophysiological role of placental-derived exosomes in pre-eclampsia. (hide)
EV-METRIC
33% (67th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Normal pregnancy (< 33 weeks gestation)
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density cushion
Filtration
Protein markers
EV: PLAP/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Normal pregnancy (< 33 weeks gestation)
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-55
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
MLA-55
Wash: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
Lowry
Western Blot
Detected EV-associated proteins
CD63
ELISA
Detected EV-associated proteins
CD63/ PLAP
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
102.9 + - 12.16
EV concentration
Yes
EV200178 3/4 Homo sapiens Blood plasma (d)(U)C
DC
Filtration
Pillay, Preenan 2016 33%

Study summary

Full title
All authors
Preenan Pillay, Niren Maharaj, Jagidesa Moodley, Irene Mackraj
Journal
Placenta
Abstract
Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biolog (show more...)Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biological cells under normal and pathological conditions. Although there have been reports of circulating exosomes in normal pregnancy, the relevance of placental-derived exosomes in normal and abnormal pregnancies still needs to be elucidated. The aim of this study was to quantify total and placental-derived exosomes in maternal plasma from normal (N), early onset- and late onset-preeclampsia (PE). Method: Plasma samples were obtained from pregnant women in the third trimester, for the isolation of exosomes by differential ultracentrifugation. Total exosomes were quantified using nanoparticle tracking analysis and immuno-reactive exosomal CD63 quantification. Placental-derived exosomes were quantified using placental alkaline phosphatase (PLAP) as a specific marker. The contribution of placental-derived exosomes to total exosomes in maternal plasma was determined by the ratio of PLAP+ exosomes to CD63+ exosomes. Results: The concentration of total exosomes significantly increased in early onset-PE and late onset-PE compared to N (≤33 weeks) and N (≥34 weeks). The relative concentration of placental-derived exosomes significantly increased in early onset-PE but decreased in late onset-PE compared to N. The ratio of PLAP+ exosomes to total number of exosomes significantly decreased in early onset-PE and late onset-PE. A positive correlation between total and placental-derived exosomes were obtained in N (≤33 weeks: Pearson's r = 0.60, ≥34 weeks: Pearson's r = 0.67) and early onset-PE (Pearson's r = 0.51, p < 0.05) with the inverse in late onset-PE (Pearson's r = -0.62, p < 0.01). Conclusion: The differences in the contribution of placental-derived exosomes to total exosomes in maternal circulation suggests a possible pathophysiological role of placental-derived exosomes in pre-eclampsia. (hide)
EV-METRIC
33% (67th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Early-onset pre-eclampsia (< 33 weeks gestation)
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density cushion
Filtration
Protein markers
EV: PLAP/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Early-onset pre-eclampsia (< 33 weeks gestation)
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-55
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
MLA-55
Wash: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
Lowry
Western Blot
Detected EV-associated proteins
CD63
ELISA
Detected EV-associated proteins
CD63/ PLAP
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
101.8 + - 7.68 nm
EV concentration
Yes
EV200178 4/4 Homo sapiens Blood plasma (d)(U)C
DC
Filtration
Pillay, Preenan 2016 33%

Study summary

Full title
All authors
Preenan Pillay, Niren Maharaj, Jagidesa Moodley, Irene Mackraj
Journal
Placenta
Abstract
Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biolog (show more...)Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biological cells under normal and pathological conditions. Although there have been reports of circulating exosomes in normal pregnancy, the relevance of placental-derived exosomes in normal and abnormal pregnancies still needs to be elucidated. The aim of this study was to quantify total and placental-derived exosomes in maternal plasma from normal (N), early onset- and late onset-preeclampsia (PE). Method: Plasma samples were obtained from pregnant women in the third trimester, for the isolation of exosomes by differential ultracentrifugation. Total exosomes were quantified using nanoparticle tracking analysis and immuno-reactive exosomal CD63 quantification. Placental-derived exosomes were quantified using placental alkaline phosphatase (PLAP) as a specific marker. The contribution of placental-derived exosomes to total exosomes in maternal plasma was determined by the ratio of PLAP+ exosomes to CD63+ exosomes. Results: The concentration of total exosomes significantly increased in early onset-PE and late onset-PE compared to N (≤33 weeks) and N (≥34 weeks). The relative concentration of placental-derived exosomes significantly increased in early onset-PE but decreased in late onset-PE compared to N. The ratio of PLAP+ exosomes to total number of exosomes significantly decreased in early onset-PE and late onset-PE. A positive correlation between total and placental-derived exosomes were obtained in N (≤33 weeks: Pearson's r = 0.60, ≥34 weeks: Pearson's r = 0.67) and early onset-PE (Pearson's r = 0.51, p < 0.05) with the inverse in late onset-PE (Pearson's r = -0.62, p < 0.01). Conclusion: The differences in the contribution of placental-derived exosomes to total exosomes in maternal circulation suggests a possible pathophysiological role of placental-derived exosomes in pre-eclampsia. (hide)
EV-METRIC
33% (67th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Late-onset pre-eclampsia (> 34 weeks gestation)
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density cushion
Filtration
Protein markers
EV: PLAP/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Late-onset pre-eclampsia (> 34 weeks gestation)
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-55
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
MLA-55
Wash: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Sucrose
Characterization: Protein analysis
Protein Concentration Method
Lowry
Western Blot
Detected EV-associated proteins
CD63
ELISA
Detected EV-associated proteins
CD63/ PLAP
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
104.1 + - 7.65 nm
EV concentration
Yes
EV200168 1/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Rh36
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Rh36
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ TSG101
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-120
EV200168 2/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Rh30
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Pax3-FOXO1/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Rh30
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ Pax3-FOXO1/ TSG101
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-120
EV200168 3/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
RD
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
RD
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
TSG101/ HSC70/ GAPDH
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-120
EV200168 4/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Rh41
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Pax3-FOXO1/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Rh41
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ Pax3-FOXO1/ TSG101
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-120
EV200168 5/5 Homo sapiens Cell culture supernatant (d)(U)C Ghayad, Sandra E 2016 33%

Study summary

Full title
All authors
Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab
Journal
Sci Rep
Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
JR1
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ GAPDH/ HSC70
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
JR1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSC70/ GAPDH/ TSG101
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.2
Characterization: Particle analysis
EM
EM-type
Scanning-EM
Image type
Wide-field
Report size (nm)
40-120
EV160012 1/8 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Hannafon BN 2016 33%

Study summary

Full title
All authors
Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, Zhao ZJ, Blick KE, Dooley WC, Ding WQ.
Journal
Breast Cancer Res Treat
Abstract
BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific (show more...)BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
MCF7
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: CD63
non-EV:
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MCF7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
2h at 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
60
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD63
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
103.3
EV concentration
Yes
EM
EM-type
Immuno-EM
Proteïns
CD63
Image type
Close-up, Wide-field
EV160012 2/8 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Hannafon BN 2016 33%

Study summary

Full title
All authors
Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, Zhao ZJ, Blick KE, Dooley WC, Ding WQ.
Journal
Breast Cancer Res Treat
Abstract
BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific (show more...)BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
MDAMB231
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: CD63
non-EV:
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MDAMB231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
2h at 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
60
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD63
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
99.6
EV concentration
Yes
EM
EM-type
Immuno-EM
Proteïns
CD63
Image type
Close-up, Wide-field
EV160011 1/4 Homo sapiens Cell culture supernatant (d)(U)C
ExoQuick
Filtration
Hoshina S 2016 33%

Study summary

Full title
All authors
Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H.
Journal
PLoS One
Abstract
Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
BCBL1
Sample origin
Kaposi sarcoma-associated herpesvirus-infected
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Filtration
Protein markers
EV: Lyn/ CD63/ HSP70
non-EV: KSHV ORF45
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
Kaposi sarcoma-associated herpesvirus-infected
EV-producing cells
BCBL1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Lyn/ HSP70
Detected contaminants
KSHV ORF45
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160011 2/4 Homo sapiens Cell culture supernatant (d)(U)C
ExoQuick
Filtration
Hoshina S 2016 33%

Study summary

Full title
All authors
Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H.
Journal
PLoS One
Abstract
Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
TY1
Sample origin
Kaposi sarcoma-associated herpesvirus-infected
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Filtration
Protein markers
EV: HSP70/ Lyn/ CD63
non-EV: KSHV ORF45
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
Kaposi sarcoma-associated herpesvirus-infected
EV-producing cells
TY1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Lyn/ HSP70
Not detected contaminants
KSHV ORF45
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160011 3/4 Homo sapiens Cell culture supernatant (d)(U)C
ExoQuick
Filtration
Hoshina S 2016 33%

Study summary

Full title
All authors
Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H.
Journal
PLoS One
Abstract
Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
LCL
Sample origin
Epstein-Barr virus-infected
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Filtration
Protein markers
EV: HSP70/ Lyn/ CD63
non-EV: KSHV ORF45
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
Epstein-Barr virus-infected
EV-producing cells
LCL
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ HSP70/ Lyn
Not detected contaminants
KSHV ORF45
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160011 4/4 Homo sapiens Cell culture supernatant (d)(U)C
ExoQuick
Filtration
Hoshina S 2016 33%

Study summary

Full title
All authors
Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H.
Journal
PLoS One
Abstract
Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Bjab
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Filtration
Protein markers
EV: HSP70/ Lyn/ CD63
non-EV: KSHV ORF45
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
Control condition
EV-producing cells
Bjab
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Lyn/ CD63/ HSP70
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160008 3/3 Rattus norvegicus Cell culture supernatant (d)(U)C Cianciaruso C 2016 33%

Study summary

Full title
All authors
Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S
Journal
Diabetes
Abstract
The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide)
EV-METRIC
33% (61st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
INS1
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alix
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
INS1
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
87.5
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: speed (g)
110000
Wash: time (min)
70
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix
Proteomics
Proteomics database
No
EV210106 1/5 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Andreu, Zoraida 2016 29%

Study summary

Full title
All authors
Zoraida Andreu, Renan Otta Oshiro, Alberto Redruello, Soraya López-Martín, Cristina Gutiérrez-Vázquez, Esperanza Morato, Ana Isabel Marina, Carlos Olivier Gómez, María Yáñez-Mó
Journal
Eur J Pharm Sci.
Abstract
Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Cu (show more...)Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. (hide)
EV-METRIC
29% (54th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
J82
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
J82
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
JS-24.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
33
Wash: time (min)
60
Wash: Rotor Type
JS-24.38
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EV210106 2/5 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Andreu, Zoraida 2016 29%

Study summary

Full title
All authors
Zoraida Andreu, Renan Otta Oshiro, Alberto Redruello, Soraya López-Martín, Cristina Gutiérrez-Vázquez, Esperanza Morato, Ana Isabel Marina, Carlos Olivier Gómez, María Yáñez-Mó
Journal
Eur J Pharm Sci.
Abstract
Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Cu (show more...)Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. (hide)
EV-METRIC
29% (54th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
UMUC-3
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
UMUC-3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
JS-24.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
33
Wash: time (min)
60
Wash: Rotor Type
JS-24.38
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EV210106 3/5 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Andreu, Zoraida 2016 29%

Study summary

Full title
All authors
Zoraida Andreu, Renan Otta Oshiro, Alberto Redruello, Soraya López-Martín, Cristina Gutiérrez-Vázquez, Esperanza Morato, Ana Isabel Marina, Carlos Olivier Gómez, María Yáñez-Mó
Journal
Eur J Pharm Sci.
Abstract
Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Cu (show more...)Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. (hide)
EV-METRIC
29% (54th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
SW780
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
SW780
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
JS-24.38
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
33
Wash: time (min)
60
Wash: Rotor Type
JS-24.38
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EV210101 1/6 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
Filtration
Welton, Joanne Louise 2016 29%

Study summary

Full title
All authors
Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton
Journal
J Extracell Vesicles
Abstract
Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide)
EV-METRIC
29% (62nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Metastatic prostate cancer, failed treatment
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Size-exclusion chromatography (non-commercial)
Filtration
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
Blood plasma
Sample Condition
Metastatic prostate cancer, failed treatment
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Other;Spectrophotometry
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
SOMAscan multiplex assay
EV210101 2/6 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
Filtration
Welton, Joanne Louise 2016 29%

Study summary

Full title
All authors
Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton
Journal
J Extracell Vesicles
Abstract
Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide)
EV-METRIC
29% (62nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Mestatatic prostate cancer, prior treatment
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Size-exclusion chromatography (non-commercial)
Filtration
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
Blood plasma
Sample Condition
Mestatatic prostate cancer, prior treatment
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Other;Spectrophotometry
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
SOMAscan multiplex assay
EV210101 5/6 Homo sapiens Urine (d)(U)C
Filtration
SEC (non-commercial)
Welton, Joanne Louise 2016 29%

Study summary

Full title
All authors
Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton
Journal
J Extracell Vesicles
Abstract
Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide)
EV-METRIC
29% (58th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Metastatic prostate cancer, failed treatment
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Size-exclusion chromatography (non-commercial)
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
Urine
Sample Condition
Metastatic prostate cancer, failed treatment
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
2.8
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Other;Spectrophotometry
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
SOMAscan multiplex assay
EV210101 6/6 Homo sapiens Urine (d)(U)C
Filtration
SEC (non-commercial)
Welton, Joanne Louise 2016 29%

Study summary

Full title
All authors
Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton
Journal
J Extracell Vesicles
Abstract
Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide)
EV-METRIC
29% (58th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Mestatatic prostate cancer, prior treatment
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Size-exclusion chromatography (non-commercial)
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
Urine
Sample Condition
Mestatatic prostate cancer, prior treatment
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
2.8
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Other;Spectrophotometry
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
SOMAscan multiplex assay
EV210036 1/2 Homo sapiens Cell culture supernatant (d)(U)C
UF
Knol, Jaco C 2016 29%

Study summary

Full title
All authors
Jaco C Knol, Inge de Reus, Tim Schelfhorst, Robin Beekhof, Meike de Wit, Sander R Piersma, Thang V Pham, Egbert F Smit, Henk M W Verheul, Connie R Jiménez
Journal
EuPA Open Proteom.
Abstract
Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived (show more...)Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizing Heat Shock Protein (HSP)-binding peptide Vn96 to aggregate HSP-decorated EVs, which can be performed at small 'miniprep' scale. Based on microscopic, immunoblot, and RNA sequencing analyses this method compared well with ultracentrifugation-mediated EV isolation, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cell-conditioned medium. Despite a 30-fold different scale (ultracentrifugation: 60 ml/Vn96-mediated aggregation: 2 ml) both methods yielded comparable numbers of identified proteins (3115/3085), with similar reproducibility of identification (72.5%/75.5%) and spectral count-based quantification (average CV: 31%/27%). EV fractions obtained with either method contained established EV markers and proteins linked to vesicle-related gene ontologies. Thus, Vn96 peptide-mediated aggregation is an advantageous, simple and rapid approach for EV isolation from small biological samples, enabling high-throughput analysis in a biomarker discovery setting. (hide)
EV-METRIC
29% (54th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HT-29
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HT-29
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
114000
Wash: volume per pellet (ml)
13.2
Wash: time (min)
90
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
114000
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Not specified
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EV210137 2/2 Homo sapiens Serum ExoQuick Youn Lee, Joo 2016 28%

Study summary

Full title
All authors
Joo Youn Lee, Jin Kyun Park, Eun Young Lee, Eun Bong Lee, Yeong Wook Song
Journal
Arthritis Res Ther
Abstract
Background: Exosomes are involved in intercellular communication. The aim of this study was to inves (show more...)Background: Exosomes are involved in intercellular communication. The aim of this study was to investigate whether circulating exosomes effectively contribute to the inflammatory response in systemic lupus erythematosus (SLE). Methods: Exosomes were purified from SLE patients and healthy controls (HCs). Healthy peripheral blood mononuclear cells (PBMCs) were stimulated with exosomes isolated from SLE patients and HCs in the presence or absence of Toll-like receptor (TLR) inhibitors. Production of interferon (IFN)-α, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were measured. Correlation between exosome levels and SLE disease activity was examined. Results: The serum exosomes levels were significantly higher in SLE patients than in HCs. SLE exosomes induced a higher production of IFN-α, TNF-α, IL-1β, and IL-6 compared to healthy exosomes. SLE serum that was depleted of exosomes and SLE exosomes that were mechanically disrupted failed to induce any significant cytokine production. Exosome-mediated production of TNF-α, IL-1β, and IL-6 was decreased by the TLR4 antagonist, whereas that of IFN-α was suppressed by the TLR1/2, TLR7, and TLR9 antagonists. Exosome levels correlated with disease activity in SLE patients (rho = 0.846, p = 0.008). Conclusions: The circulating exosomes are immunologically active and their levels correlate with disease activity in SLE patients. The circulating exosomes might serve as novel biomarkers of SLE disease activity. (hide)
EV-METRIC
28% (74th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Systemic lupus erythematosus
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: CD81/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Systemic lupus erythematosus
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Not determined
ELISA
Detected EV-associated proteins
CD63/ CD81
Flow cytometry
Hardware adjustments
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV210136 1/2 Pseudomonas maltophilia Cell culture supernatant (d)(U)C
DG
Filtration
Ferrer-Navarro, Mario 2016 28%

Study summary

Full title
All authors
Mario Ferrer-Navarro, Gerard Torrent, Elías Mongiardini, Oscar Conchillo-Solé, Isidre Gibert, Xavier Daura
Journal
J Proteomics
Abstract
Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that dis (show more...)Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that displays a high genomic diversity, complicating the study of its pathogenicity, virulence and resistance factors. The interaction of bacterial pathogens with host cells is largely mediated by outer membrane proteins (OMPs). Indeed, several OMPs of Gram-negative bacteria have been recognized as important virulence factors and targets for host immune recognition or to be involved in mechanisms of resistance to antimicrobials. OMPs are also present in outer membrane vesicles (OMVs), which bacteria constitutively secrete to the extracellular milieu and are essential for bacterial survival and pathogenesis. Here, we report the characterization of the OMP and native OMV subproteomes of a clinical isolate (M30) and a collection strain (ATCC13637) of S. maltophilia. We had previously shown that the ATCC13637 strain has an attenuated phenotype in a zebrafish model of infection, as well as a distinct susceptibility profile against a panel of antimicrobials. The protein profiles of the OMP and OMV subproteomes of these two strains and their differences consequently point at pathogenesis, virulence or resistance proteins, such as two variants of the quorum-sensing factor Ax21 that are found to be highly abundant in the OMP fraction and exported to OMVs. Biological significance: Stenotrophomonas maltophilia is rapidly climbing positions in the ranking of multidrug-resistant pathogens that are frequently isolated in hospital environments. Being an emerging human pathogen, the knowledge on the factors determining the pathogenicity, virulence and resistance traits of this microorganism is still scarce. Outer membrane proteins (OMPs) and vesicles (OMVs) are key elements for the interaction of Gram-negative bacteria with their environment -including the host-and have fundamental roles in both infection and resistance processes. The present study sets a first basis for a phenotype-dependent characterisation of the OMP subproteome of S. maltophilia and complements very recent work on the OMV subproteome of this species. The variability found among even two strains demonstrates once more that the analysis of genotypically and phenotypically distinct isolates under various conditions will be required before we can draw a significant picture of the OMP and OMV subproteomes of S. maltophilia. (hide)
EV-METRIC
28% (52nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Pseudomonas maltophilia
Sample origin
ATCC 13637
Focus vesicles
Outer membrane vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
Not specified
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Pseudomonas maltophilia
Sample Type
Cell culture supernatant
Sample Condition
ATCC 13637
EV-producing cells
Pseudomonas maltophilia
EV-harvesting Medium
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
150000
Density gradient
Density medium
Sucrose
Type
Discontinous
Number of initial discontinuous layers
3
Lowest density fraction
0.6 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
1,25
Orientation
Top-down
Rotor type
Not specified
Speed (g)
200000
Duration (min)
1200
Fraction volume (mL)
Not specified
Fraction processing
Centrifugation
Pelleting: volume per fraction
Not spec
Pelleting: duration (min)
120
Pelleting: rotor type
Not specified
Pelleting: speed (g)
200000
Filtration steps
0.45µm > x > 0.22µm, No
Characterization: Protein analysis
Protein Concentration Method
2D-Quant kit
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Wide-field
EV210136 2/2 Pseudomonas maltophilia Cell culture supernatant (d)(U)C
DG
Filtration
Ferrer-Navarro, Mario 2016 28%

Study summary

Full title
All authors
Mario Ferrer-Navarro, Gerard Torrent, Elías Mongiardini, Oscar Conchillo-Solé, Isidre Gibert, Xavier Daura
Journal
J Proteomics
Abstract
Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that dis (show more...)Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that displays a high genomic diversity, complicating the study of its pathogenicity, virulence and resistance factors. The interaction of bacterial pathogens with host cells is largely mediated by outer membrane proteins (OMPs). Indeed, several OMPs of Gram-negative bacteria have been recognized as important virulence factors and targets for host immune recognition or to be involved in mechanisms of resistance to antimicrobials. OMPs are also present in outer membrane vesicles (OMVs), which bacteria constitutively secrete to the extracellular milieu and are essential for bacterial survival and pathogenesis. Here, we report the characterization of the OMP and native OMV subproteomes of a clinical isolate (M30) and a collection strain (ATCC13637) of S. maltophilia. We had previously shown that the ATCC13637 strain has an attenuated phenotype in a zebrafish model of infection, as well as a distinct susceptibility profile against a panel of antimicrobials. The protein profiles of the OMP and OMV subproteomes of these two strains and their differences consequently point at pathogenesis, virulence or resistance proteins, such as two variants of the quorum-sensing factor Ax21 that are found to be highly abundant in the OMP fraction and exported to OMVs. Biological significance: Stenotrophomonas maltophilia is rapidly climbing positions in the ranking of multidrug-resistant pathogens that are frequently isolated in hospital environments. Being an emerging human pathogen, the knowledge on the factors determining the pathogenicity, virulence and resistance traits of this microorganism is still scarce. Outer membrane proteins (OMPs) and vesicles (OMVs) are key elements for the interaction of Gram-negative bacteria with their environment -including the host-and have fundamental roles in both infection and resistance processes. The present study sets a first basis for a phenotype-dependent characterisation of the OMP subproteome of S. maltophilia and complements very recent work on the OMV subproteome of this species. The variability found among even two strains demonstrates once more that the analysis of genotypically and phenotypically distinct isolates under various conditions will be required before we can draw a significant picture of the OMP and OMV subproteomes of S. maltophilia. (hide)
EV-METRIC
28% (52nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Pseudomonas maltophilia
Sample origin
Clinical strain M30
Focus vesicles
Outer membrane vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
Not specified
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Pseudomonas maltophilia
Sample Type
Cell culture supernatant
Sample Condition
Clinical strain M30
EV-producing cells
Pseudomonas maltophilia
EV-harvesting Medium
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
150000
Density gradient
Density medium
Sucrose
Type
Discontinous
Number of initial discontinuous layers
3
Lowest density fraction
0.6 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
1,25
Orientation
Top-down
Rotor type
Not specified
Speed (g)
200000
Duration (min)
1200
Fraction volume (mL)
Not specified
Fraction processing
Centrifugation
Pelleting: volume per fraction
Not spec
Pelleting: duration (min)
120
Pelleting: rotor type
Not specified
Pelleting: speed (g)
200000
Filtration steps
0.45µm > x > 0.22µm, No
Characterization: Protein analysis
Protein Concentration Method
2D-Quant kit
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Wide-field
EV210099 3/9 Homo sapiens Cell culture supernatant ExoQuick Rider, Mark A 2016 25%

Study summary

Full title
All authors
Mark A Rider, Stephanie N Hurwitz, David G Meckes Jr
Journal
Sci Rep
Abstract
Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known (show more...)Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes. (hide)
EV-METRIC
25% (51st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK293 T
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: CD63/ TSG101/ HSP70/ Alix
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development/ Technical analysis comparing/optimizing EV-­related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HEK293 T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ HSP70/ Alix
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Not Reported
Particle yield
NA NA
EV210099 4/9 Homo sapiens Cell culture supernatant Total Exosome Isolation Rider, Mark A 2016 25%

Study summary

Full title
All authors
Mark A Rider, Stephanie N Hurwitz, David G Meckes Jr
Journal
Sci Rep
Abstract
Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known (show more...)Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes. (hide)
EV-METRIC
25% (51st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK293 T
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Total Exosome Isolation
Protein markers
EV: CD63/ TSG101/ HSP70/ Alix
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development/ Technical analysis comparing/optimizing EV-­related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HEK293 T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ HSP70/ Alix
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Not Reported
Particle yield
NA NA
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
Not specified
EV210080 1/2 Homo sapiens Serum (d)(U)C
ExoQuick
Shen, Rong-Kai 2016 25%

Study summary

Full title
All authors
Rong-Kai Shen, Xia Zhu, Huan Yi, Chao-Yang Wu, Fei Chen, Li-Qun Dai, Jian-Hua Lin
Journal
Int J Clin Exp Pathol
Abstract
http://www.ijcep.com/files/ijcep0019652.pdf Osteosarcoma (OS) is the most common primary bone tumo (show more...)http://www.ijcep.com/files/ijcep0019652.pdf Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Combined therapy has not improved the prognosis of osteosarcoma in the past five years, while new strategies need to be explored. We extracted serum exosomes from OS and healthy people. Contrasting to exosomes from healthy ones, those derived from patients suffering from OS significantly promoted the ability of adhesion, migration and viability of the MG63 in vitro study. Osteosarcoma-derived exosomes package proteins were mainly intracellular ones, while these proteins exclusively expressed glucose-6-phospate dehydrogenase (G6PD), phosphofructokinase, transaldolase 1 and transketolase that reprogrammed tumor metabolism and prompted progression of osteosarcoma. Western-blot assay confirmed osteosarcoma-derived exosomes contained generous G6PD than health people. In conclusion, serum exosomal proteins originated from osteosarcoma and healthy people were quite different from each other. Exosomes derived from osteosarcoma could promote tumor progression, for G6PD were largely packaged by serum exosomes. These special serum exosomes, which are abundant in G6PD, can be a promising target for diagnostic and therapeutic strategy for osteosarcoma. (hide)
EV-METRIC
25% (71st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Protein markers
EV: CD63/ Actin/ G6PD
non-EV: None
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ Actin/ G6PD
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
30-100
EV210080 2/2 Homo sapiens Serum (d)(U)C
ExoQuick
Shen, Rong-Kai 2016 25%

Study summary

Full title
All authors
Rong-Kai Shen, Xia Zhu, Huan Yi, Chao-Yang Wu, Fei Chen, Li-Qun Dai, Jian-Hua Lin
Journal
Int J Clin Exp Pathol
Abstract
http://www.ijcep.com/files/ijcep0019652.pdf Osteosarcoma (OS) is the most common primary bone tumo (show more...)http://www.ijcep.com/files/ijcep0019652.pdf Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Combined therapy has not improved the prognosis of osteosarcoma in the past five years, while new strategies need to be explored. We extracted serum exosomes from OS and healthy people. Contrasting to exosomes from healthy ones, those derived from patients suffering from OS significantly promoted the ability of adhesion, migration and viability of the MG63 in vitro study. Osteosarcoma-derived exosomes package proteins were mainly intracellular ones, while these proteins exclusively expressed glucose-6-phospate dehydrogenase (G6PD), phosphofructokinase, transaldolase 1 and transketolase that reprogrammed tumor metabolism and prompted progression of osteosarcoma. Western-blot assay confirmed osteosarcoma-derived exosomes contained generous G6PD than health people. In conclusion, serum exosomal proteins originated from osteosarcoma and healthy people were quite different from each other. Exosomes derived from osteosarcoma could promote tumor progression, for G6PD were largely packaged by serum exosomes. These special serum exosomes, which are abundant in G6PD, can be a promising target for diagnostic and therapeutic strategy for osteosarcoma. (hide)
EV-METRIC
25% (71st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Osteosarcoma
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Protein markers
EV: CD63/ Actin/ G6PD
non-EV: None
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Osteosarcoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ Actin/ G6PD
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
No
EV210078 3/5 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
ExoQuick
Macklin, Rebecca 2016 25%

Study summary

Full title
All authors
Rebecca Macklin, Haolu Wang, Dorothy Loo, Sally Martin, Andrew Cumming, Na Cai, Rebecca Lane, Natalia Saenz Ponce, Eleni Topkas, Kerry Inder, Nicholas A Saunders, Liliana Endo-Munoz
Journal
Oncotarget
Abstract
Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pu (show more...)Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs. (hide)
EV-METRIC
25% (51st 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