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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in isolation protocol/particle analysis/sample origin/sample type
Experiment number
  • Experiments differ in Isolation, Proteïn analysis, Particle analysis
Experiment number
  • Experiments differ in Sample type, Separation protocol
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  • Experiments differ in isolation protocol/particle analysis/sample origin/sample type
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  • Experiments differ in Sample type
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Experiment number
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Experiment number
  • Experiments differ in Sample condition, Separation protocol
Experiment number
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Experiment number
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Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV180059 5/6 Gut microbiota Blood plasma SEC Tulkens J 2018 42%

Study summary

Full title
All authors
Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A.
Journal
Gut
Abstract
(show more...) (hide)
EV-METRIC
42% (83rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Therapy-induced intestinal mucositis
Focus vesicles
LPS-positive bacterial EV
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
SEC
Protein markers
EV: LPS/ OmpA
non-EV:
Proteomics
no
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Gut microbiota
Sample Type
Blood plasma
Sample Condition
Therapy-induced intestinal mucositis
Separation Method
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
No
Detected EV-associated proteins
LPS
Characterization: Particle analysis
EM
EM-type
Immuno-EM
Proteïns
E. coli LPS
Image type
Close-up
EV180004 1/1 Homo sapiens Blood plasma (d)(U)C
qEV
Picciolini S 2018 37%

Study summary

Full title
All authors
Picciolini S, Gualerzi A, Vanna R, Sguassero A, Gramatica F, Bedoni M, Masserini M, Morasso C
Journal
Anal Chem
Abstract
The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealin (show more...)The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases. (hide)
EV-METRIC
37% (79th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
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
(d)(U)C + qEV
Protein markers
EV: CD81/ ephrinB/ CD171/ PLP1/ Flotillin-1/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
59.77
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD81, Flotillin-1
Other 1
Surface plasmon resonance imaging
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
1500000000
EM
EM-type
Transmission-EM
Image type
Wide-field
EV170014 3/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
SEC
Dionysios C Watson 2018 37%

Study summary

Full title
All authors
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
Journal
J Extracell Vesicles
Abstract
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide)
EV-METRIC
37% (70th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration + SEC
Protein markers
EV: hetIL-15/Lactadherin
non-EV: ferritin
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
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
EV-producing cells
HEK293
EV-harvesting Medium
Serum free medium
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
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
5
Resin type
Superdex 200
Characterization: Protein analysis
Protein Concentration Method
Bradford
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
Particle yield
240000000000
EV180026 2/7 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Wenzhe Li 2018 33%

Study summary

Full title
All authors
Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang
Journal
Small methods
Abstract
Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: CD81/ Flotillin-1/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MCF10A
EV-harvesting Medium
EV-depleted serum
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 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63, CD81, Flotillin-1
Not detected contaminants
Calnexin
EV180026 4/7 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Wenzhe Li 2018 33%

Study summary

Full title
All authors
Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang
Journal
Small methods
Abstract
Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: CD81/ Flotillin-1/ CD63/ EpCAM
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MDAMB468
EV-harvesting Medium
EV-depleted serum
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 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63, CD81, Flotillin-1, EpCAM
Not detected contaminants
Calnexin
EV180026 6/7 Homo sapiens Serum (d)(U)C
Filtration
Wenzhe Li 2018 33%

Study summary

Full title
All authors
Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang
Journal
Small methods
Abstract
Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide)
EV-METRIC
33% (79th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Adj. k-factor
104.6 (pelleting) / 104.6 (washing)
Protein markers
EV: CD81/ Flotillin-1/ CD63/ EpCAM
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
1200
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
150000
Pelleting: adjusted k-factor
104.6
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
150000
Wash: adjusted k-factor
104.6
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63, CD81, Flotillin-1
Not detected contaminants
Albumin
EV180030 2/7 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Zhaohao Liao 2018 33%

Study summary

Full title
All authors
Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + qEV
Protein markers
EV: / CD81/ CD63
non-EV: AChE
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
H9
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 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ CD81
Other 1
Acetylcholinesterase assay
Detected EV-associated proteins
Detected contaminants
AChE
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV180030 1/7 Homo sapiens Cell culture supernatant (d)(U)C Zhaohao Liao 2018 29%

Study summary

Full title
All authors
Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV:
non-EV: AChE
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
U937
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 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Protein Concentration Method
Not determined
Other 1
Acetylcholinesterase assay
Detected EV-associated proteins
Detected contaminants
AChE
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV180030 4/7 Homo sapiens Cell culture supernatant (d)(U)C Zhaohao Liao 2018 29%

Study summary

Full title
All authors
Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV:
non-EV: AChE
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PM1
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 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Protein Concentration Method
Not determined
Other 1
Acetycholinesterase assay
Detected EV-associated proteins
Detected contaminants
AChE
EV180059 4/6 Gut microbiota Blood plasma SEC Tulkens J 2018 28%

Study summary

Full title
All authors
Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A.
Journal
Gut
Abstract
(show more...) (hide)
EV-METRIC
28% (69th 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
HIV
Focus vesicles
LPS-positive bacterial EV
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
SEC
Protein markers
EV: LPS/ OmpA
non-EV:
Proteomics
no
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Gut microbiota
Sample Type
Blood plasma
Sample Condition
HIV
Separation Method
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
No
Detected EV-associated proteins
LPS
EV170037 1/3 Homo sapiens Serum (d)(U)C Klump, Jennifer 2018 28%

Study summary

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

Study summary

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

Study summary

Full title
All authors
Klump J, Phillipp U, Follo M, Eremin A, Lehmann H, Nestel S, von Bubnoff N, Nazarenko I
Journal
Nanomedicine
Abstract
Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood bi (show more...)Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. (hide)
EV-METRIC
28% (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
Mastocytosis
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Adj. k-factor
213.2 (pelleting) / 213.2 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Mastocytosis
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
120
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
213.2
Protein Concentration Method
microBCA
Protein Concentration
10-50 dependent whether healthy donors or cancer patients were analyzed
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
different populations of vesicles were detected by DLS and NTA;120-500 nm; and over 1000 nm
NTA
Report type
Size range/distribution
Reported size (nm)
90-300
EV concentration
Yes
Particle yield
3.00E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
90-120
Extra information
Publication aimed to determine the content of mutated DNA oncogenes copy inside of the vesicles (post- DNase treatments) and in the free-circulating fractions. For that DNA was isolated from different EV and fc fractions and DNA was analyzed using ddPCR. Conclusion was that the mutated BRAF and c-KIT copies are preferably located in the free-circulating fractions and not in EVs.
EV180030 7/7 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Zhaohao Liao 2018 22%

Study summary

Full title
All authors
Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide)
EV-METRIC
22% (47th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + qEV
Protein markers
EV: AChE/ CD63
non-EV:
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary red blood cells
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 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63
Other 1
Acetylcholinesterase assay
Detected EV-associated proteins
AChE
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV180001 1/2 Homo sapiens Serum Exiqon miRCURY exosome isolation kit
Filtration
Kwok HH 2018 12%

Study summary

Full title
All authors
Kwok HH, Ning Z, Chong PW, Wan TS, Ng MH, Ho GYF, Ip MS, Lam DC
Journal
Cancer (basel)
Abstract
Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcino (show more...)Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcinoma. Nonetheless tumour consists of heterogeneous cell subpopulations with diverse phenotypes and genotypes, and cancer cells can actively release extracellular vesicles (EVs) to modulate the phenotype of other cells in the tumour microenvironment. We hypothesized that EVs derived from a drug-resistant subpopulation of cells could induce drug resistance in recipient cells. We have established ALK-translocated lung adenocarcinoma cell lines and subclones. The subclones have been characterized and the expression of EV-RNAs determined by quantitative polymerase chain reaction. The effects of EV transfer on drug resistance were examined in vitro. Serum EV-RNA was assayed serially in two patients prescribed ALK-tyrosine kinase inhibitor (ALK-TKI) treatment. We demonstrated that the EVs from an ALK-TKI-resistant subclone could induce drug resistance in the originally sensitive subclone. EV-RNA profiling revealed that miRNAs miR-21-5p and miR-486-3p, and lncRNAs MEG3 and XIST were differentially expressed in the EVs secreted by the resistant subclones. These circulating EV-RNA levels have been found to correlate with disease progression of EML4-ALK-translocated lung adenocarcinoma in patients prescribed ALK-TKI treatment. The results from this study suggest that EVs released by a drug-resistant subpopulation can induce drug resistance in other subpopulations and may sustain intratumoural heterogeneity. (hide)
EV-METRIC
12% (47th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Exiqon miRCURY exosome isolation kit + Filtration
Protein markers
EV: CD63
non-EV: GP96
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
Exiqon miRCURY exosome isolation kit
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63
Not detected contaminants
GP96
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
84
EV180001 2/2 Homo sapiens Cell culture supernatant Exiqon miRCURY exosome isolation kit
Filtration
Kwok HH 2018 12%

Study summary

Full title
All authors
Kwok HH, Ning Z, Chong PW, Wan TS, Ng MH, Ho GYF, Ip MS, Lam DC
Journal
Cancer (basel)
Abstract
Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcino (show more...)Anaplastic lymphoma kinase (ALK) translocation is an actionable mutation in lung adenocarcinoma. Nonetheless tumour consists of heterogeneous cell subpopulations with diverse phenotypes and genotypes, and cancer cells can actively release extracellular vesicles (EVs) to modulate the phenotype of other cells in the tumour microenvironment. We hypothesized that EVs derived from a drug-resistant subpopulation of cells could induce drug resistance in recipient cells. We have established ALK-translocated lung adenocarcinoma cell lines and subclones. The subclones have been characterized and the expression of EV-RNAs determined by quantitative polymerase chain reaction. The effects of EV transfer on drug resistance were examined in vitro. Serum EV-RNA was assayed serially in two patients prescribed ALK-tyrosine kinase inhibitor (ALK-TKI) treatment. We demonstrated that the EVs from an ALK-TKI-resistant subclone could induce drug resistance in the originally sensitive subclone. EV-RNA profiling revealed that miRNAs miR-21-5p and miR-486-3p, and lncRNAs MEG3 and XIST were differentially expressed in the EVs secreted by the resistant subclones. These circulating EV-RNA levels have been found to correlate with disease progression of EML4-ALK-translocated lung adenocarcinoma in patients prescribed ALK-TKI treatment. The results from this study suggest that EVs released by a drug-resistant subpopulation can induce drug resistance in other subpopulations and may sustain intratumoural heterogeneity. (hide)
EV-METRIC
12% (30th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Exiqon miRCURY exosome isolation kit + Filtration
Protein markers
EV: CD63
non-EV: GP96
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
FA34
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Commercial EDS
Separation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
Exiqon miRCURY exosome isolation kit
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Concentration
50
Western Blot
Detected EV-associated proteins
CD63
Not detected contaminants
GP96
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
109
EV200104 1/3 Homo sapiens Cerebrospinal fluid miRCURY Exosome Isolation Kit (Exiqon) Paul M. McKeever 2018 0%

Study summary

Full title
All authors
Paul M. McKeever, Raphael Schneider, Foad Taghdiri, Anna Weichert, Namita Multani, Robert A. Brown, Adam L. Boxer, Anna Karydas, Bruce Miller, Janice Robertson, Maria Carmela Tartaglia
Journal
Molecular Neurobiology
Abstract
Clinical diagnosis of Alzheimer’s disease (AD) prior to the age of 65 years is classified as young (show more...)Clinical diagnosis of Alzheimer’s disease (AD) prior to the age of 65 years is classified as young-onset (YOAD), whereas diagnosis after the age of 65 years is considered late-onset (LOAD). Although rare autosomal mutations more commonly associate with YOAD, most YOAD and LOAD cases are sporadic. YOAD and LOAD share amyloid and tau pathology, but many YOAD patients show increased disease severity and rate of progression. The current study examined the microRNA (miRNA) expression profile from exosomes isolated from the cerebrospinal fluid (CSF) of YOAD patients with biomarkerconfirmed AD. Results uncovered miR-16-5p, miR-125b-5p, miR-451a, and miR-605-5p as differentially expressed in the CSFderived exosomes of YOAD patients when compared with healthy controls (HC). In a cohort of LOAD patients, miR-125b-5p, miR-451a, and miR-605-5p were similarly altered in expression, but miR-16-5p showed similar expression to control. Analysis of the mRNA targets of these miRNAs revealed transcripts enriched in biological processes relevant to the post-mortem posterior cingulate cortex transcriptome in YOAD from a previously published microarray study, including those related to neuron projections, synaptic signaling, metabolism, apoptosis, and the immune system. Hence, these miRNAs represent novel targets for uncovering disease mechanisms and for biomarker development in both YOAD and LOAD. (hide)
EV-METRIC
0% (median: 0% 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
Cerebrospinal fluid
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
miRCURY Exosome Isolation Kit (Exiqon)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cerebrospinal fluid
Sample Condition
Control condition
Separation Method
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
EV200104 2/3 Homo sapiens Cerebrospinal fluid miRCURY Exosome Isolation Kit (Exiqon) Paul M. McKeever 2018 0%

Study summary

Full title
All authors
Paul M. McKeever, Raphael Schneider, Foad Taghdiri, Anna Weichert, Namita Multani, Robert A. Brown, Adam L. Boxer, Anna Karydas, Bruce Miller, Janice Robertson, Maria Carmela Tartaglia
Journal
Molecular Neurobiology
Abstract
Clinical diagnosis of Alzheimer’s disease (AD) prior to the age of 65 years is classified as young (show more...)Clinical diagnosis of Alzheimer’s disease (AD) prior to the age of 65 years is classified as young-onset (YOAD), whereas diagnosis after the age of 65 years is considered late-onset (LOAD). Although rare autosomal mutations more commonly associate with YOAD, most YOAD and LOAD cases are sporadic. YOAD and LOAD share amyloid and tau pathology, but many YOAD patients show increased disease severity and rate of progression. The current study examined the microRNA (miRNA) expression profile from exosomes isolated from the cerebrospinal fluid (CSF) of YOAD patients with biomarkerconfirmed AD. Results uncovered miR-16-5p, miR-125b-5p, miR-451a, and miR-605-5p as differentially expressed in the CSFderived exosomes of YOAD patients when compared with healthy controls (HC). In a cohort of LOAD patients, miR-125b-5p, miR-451a, and miR-605-5p were similarly altered in expression, but miR-16-5p showed similar expression to control. Analysis of the mRNA targets of these miRNAs revealed transcripts enriched in biological processes relevant to the post-mortem posterior cingulate cortex transcriptome in YOAD from a previously published microarray study, including those related to neuron projections, synaptic signaling, metabolism, apoptosis, and the immune system. Hence, these miRNAs represent novel targets for uncovering disease mechanisms and for biomarker development in both YOAD and LOAD. (hide)
EV-METRIC
0% (median: 0% 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
Cerebrospinal fluid
Sample origin
Young Onset Alzheimer Disease
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
miRCURY Exosome Isolation Kit (Exiqon)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cerebrospinal fluid
Sample Condition
Young Onset Alzheimer Disease
Separation Method
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
EV200104 3/3 Homo sapiens Cerebrospinal fluid miRCURY Exosome Isolation Kit (Exiqon) Paul M. McKeever 2018 0%

Study summary

Full title
All authors
Paul M. McKeever, Raphael Schneider, Foad Taghdiri, Anna Weichert, Namita Multani, Robert A. Brown, Adam L. Boxer, Anna Karydas, Bruce Miller, Janice Robertson, Maria Carmela Tartaglia
Journal
Molecular Neurobiology
Abstract
Clinical diagnosis of Alzheimer’s disease (AD) prior to the age of 65 years is classified as young (show more...)Clinical diagnosis of Alzheimer’s disease (AD) prior to the age of 65 years is classified as young-onset (YOAD), whereas diagnosis after the age of 65 years is considered late-onset (LOAD). Although rare autosomal mutations more commonly associate with YOAD, most YOAD and LOAD cases are sporadic. YOAD and LOAD share amyloid and tau pathology, but many YOAD patients show increased disease severity and rate of progression. The current study examined the microRNA (miRNA) expression profile from exosomes isolated from the cerebrospinal fluid (CSF) of YOAD patients with biomarkerconfirmed AD. Results uncovered miR-16-5p, miR-125b-5p, miR-451a, and miR-605-5p as differentially expressed in the CSFderived exosomes of YOAD patients when compared with healthy controls (HC). In a cohort of LOAD patients, miR-125b-5p, miR-451a, and miR-605-5p were similarly altered in expression, but miR-16-5p showed similar expression to control. Analysis of the mRNA targets of these miRNAs revealed transcripts enriched in biological processes relevant to the post-mortem posterior cingulate cortex transcriptome in YOAD from a previously published microarray study, including those related to neuron projections, synaptic signaling, metabolism, apoptosis, and the immune system. Hence, these miRNAs represent novel targets for uncovering disease mechanisms and for biomarker development in both YOAD and LOAD. (hide)
EV-METRIC
0% (median: 0% 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
Cerebrospinal fluid
Sample origin
Late Onset Alzheimer Disease
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
miRCURY Exosome Isolation Kit (Exiqon)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cerebrospinal fluid
Sample Condition
Late Onset Alzheimer Disease
Separation Method
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
EV180022 2/4 Homo sapiens Cell culture supernatant miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark) Kaur S 2018 0%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
adipose tissue-derived mesenchymal stem cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
80
Separation Method
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)
Protein Concentration Method
Not determined
EV170015 1/2 Homo sapiens Cell culture supernatant 50% PEG-8000, 0.5M NaCl, mixed with the conditioned medium 1:5 v/v respectively and incubated overnight at 4°C. The mixtures were centrifuged at 1500g for 30 minutes to pellet the EVs.
(d)(U)C
Filtration
Tabak, Saray 2018 0%

Study summary

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

Study summary

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