Search > Results

You searched for: 2017 (Year of publication)

Showing 1 - 47 of 47

Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in isolation protocol
Experiment number
  • Experiments differ in isolation protocol
Experiment number
  • Experiments differ in isolation protocol
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample condition
Experiment number
  • Experiments differ in Sample condition
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample origin
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Particle analysis
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV170008 1/3 Homo sapiens Cell culture supernatant DG
dUC
Mercedes Tkach 2017 100%

Study summary

Full title
All authors
Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C
Journal
EMBO J
Abstract
Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Protein markers
EV: CD9/ MHC2/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
EV functional activity
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary monocyte derived dendritic cell
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
90
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting: time(min)
20
Pelleting: rotor type
Swinging bucket
Pelleting: speed (g)
2000
Wash: time (min)
20
Wash: Rotor Type
Eppendorf 5810R cf; swinging bucket
Wash: speed (g)
2000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0.1
Highest density fraction
0.3
Sample volume (mL)
1.2
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 55 Ti
Speed (g)
350000
Duration (min)
60
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.5
Pelleting: duration (min)
30
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
65.69
EV-subtype
Distinction between multiple subtypes
Centrifugation steps: 2K, 10K, 100K
PMID previous EV protein analysis
26858453
Extra characterization
Protein Concentration Method
microBCA
Protein Concentration
2.9+-0.3
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9,MHC2
Flow cytometry
Type of Flow cytometry
MACSQuant Miltenyi
Calibration bead size
0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9
Characterization: Particle analysis
PMID previous EV particle analysis
26858453
Extra particle analysis
NTA
Report type
Size range/distribution
EV concentration
Yes
Particle yield
6.52E10+-2.03E10 particles/million cells
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV170008 2/3 Homo sapiens Cell culture supernatant DG
dUC
Mercedes Tkach 2017 100%

Study summary

Full title
All authors
Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C
Journal
EMBO J
Abstract
Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
2097 (pelleting) / 2097 (washing)
Protein markers
EV: CD9/ MHC2/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
EV functional activity
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary monocyte derived dendritic cell
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
90
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
40
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
10000
Pelleting: adjusted k-factor
2097.
Wash: time (min)
40
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
10000
Wash: adjusted k-factor
2097.
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0.1
Highest density fraction
0.3
Sample volume (mL)
1.2
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 55 Ti
Speed (g)
350000
Duration (min)
60
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.5
Pelleting: duration (min)
30
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
65.69
EV-subtype
Distinction between multiple subtypes
Centrifugation steps: 2K, 10K, 100K
PMID previous EV protein analysis
26858453
Extra characterization
Protein Concentration Method
microBCA
Protein Concentration
1.7+-0.3
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9,MHC2
Flow cytometry
Type of Flow cytometry
MACSQuant Miltenyi
Calibration bead size
0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9
Characterization: Particle analysis
PMID previous EV particle analysis
26858453
Extra particle analysis
NTA
Report type
Size range/distribution
EV concentration
Yes
Particle yield
4.95E10+-3.81E10 particles/million cells
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV170008 3/3 Homo sapiens Cell culture supernatant DG
dUC
Mercedes Tkach 2017 100%

Study summary

Full title
All authors
Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C
Journal
EMBO J
Abstract
Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
209.7 (pelleting) / 209.7 (washing)
Protein markers
EV: CD9/ MHC2/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
EV functional activity
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary monocyte derived dendritic cell
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
90
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
209.7
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
209.7
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0.1
Highest density fraction
0.3
Sample volume (mL)
1.2
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 55 Ti
Speed (g)
350000
Duration (min)
60
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.5
Pelleting: duration (min)
30
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
65.69
EV-subtype
Distinction between multiple subtypes
Centrifugation steps: 2K, 10K, 100K
PMID previous EV protein analysis
26858453
Extra characterization
Protein Concentration Method
microBCA
Protein Concentration
1.1+-0.2
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9,MHC2
Flow cytometry
Type of Flow cytometry
MACSQuant Miltenyi
Calibration bead size
0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9
Characterization: Particle analysis
PMID previous EV particle analysis
26858453
Extra particle analysis
NTA
Report type
Size range/distribution
EV concentration
Yes
Particle yield
3.76E10+-1.41E10 particles/million cells
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV170001 1/1 Homo sapiens Cell culture supernatant DG
dUC
UF
Vergauwen, Glenn 2017 100%

Study summary

Full title
All authors
Vergauwen G, Dhondt B, Van Deun J, De Smedt E, Berx G, Timmerman E, Gevaert K, Miinalainen I, Cocquyt V, Braems G, Van den Broecke R, Denys H, De Wever O, Hendrix A.
Journal
Sci Rep
Abstract
Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust is (show more...)Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + UF
Protein markers
EV: Alix/ CD81/ TSG101
non-EV: Argonaute-2/ PMP70
Proteomics
no
EV density (g/ml)
1.094
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF7 Rab27b-GFP
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm,
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,…)
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD81/ TSG101
Not detected contaminants
Argonaute-2/ PMP70
Characterization: Particle analysis
EV170016 1/2 Mus musculus Cell culture supernatant DG
Filtration
dUC
Ying, Wei 2017 88%

Study summary

Full title
All authors
Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, Ofrecio JM, Wollam J, Hernandez-Carretero A, Fu W, Li P, Olefsky JM
Journal
Cell
Abstract
MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we (show more...)MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis. (hide)
EV-METRIC
88% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + Filtration + dUC
Adj. k-factor
256 (pelleting) / 256 (washing)
Protein markers
EV: CD9/ CD63/ HSP70/ TSG101/ Syntenin1
non-EV: Grp94
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
adipose tissue-derived macrophages
EV-harvesting Medium
EV-depleted serum
Origin
Obesitas
Preparation of EDS
Commercial EDS
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
240-360
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
256.0
Wash: time (min)
20
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
256.0
Density gradient
Only used for validation of main results
Yes
Density medium
156.9
Type
Discontinuous
Lowest density fraction
0.1
Highest density fraction
0.3
Sample volume (mL)
0.3
Orientation
Bottom-up (sample migrates upwards)
Rotor type
Type 70 Ti
Speed (g)
350000
Duration (min)
60
Fraction volume (mL)
2.4
Fraction processing
Centrifugation
Pelleting: volume per fraction
2
Pelleting: duration (min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Pelleting-wash: volume per pellet (mL)
24
Pelleting-wash: duration (min)
30
Pelleting-wash: rotor type
156.9
Pelleting-wash: speed (g)
Type 70 Ti
Pelleting-wash: adjusted k-factor
156.9
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
1.13-1.15 g/ml
Used subtypes
Yes
Characterization: Protein analysis
Protein Concentration Method
DC protein assay
Protein Concentration
9-May
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, HSP70, TSG101, Syntenin1
Not detected contaminants
Grp94
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-150
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
50-200 nm
Extra information
Full UC and density gradient protocols not in original article
EV170004 1/1 Homo sapiens Adipose tissue DG
dUC
Filtration
Jeurissen S 2017 88%

Study summary

Full title
All authors
Jeurissen S, Vergauwen G, Van Deun J, Lapeire L, Depoorter V, Miinalainen I, Sormunen R, Van den Broecke R, Braems G, Cocquyt V, Denys H, Hendrix A
Journal
Cell Adh Migr
Abstract
Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to (show more...)Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076-1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active. (hide)
EV-METRIC
88% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Adipose tissue
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Adj. k-factor
138.6 (pelleting) / 138.6 (washing)
Protein markers
EV: CD9/ Flotillin-1/ HSP70/ FABP4/ CD63
non-EV: Calreticulin/ GM130/ Prohibitin
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Adipose tissue
Origin
breast cancer
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
138.6
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
138.6
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
1
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
297.9
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1, HSP70, FABP4
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
116
EV concentration
Yes
Particle yield
2.2 10E09
EM
EM-type
Immune-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
EV170017 1/1 Mus musculus Bronchoalveolar lavage fluid dUC Maroto, Rosario 2017 75%

Study summary

Full title
All authors
Maroto R, Zhao Y, Jamaluddin M, Popov VL, Wang H, Kalubowilage M, Zhang Y, Luisi J, Sun H, Culbertson CT, Bossmann SH, Motamedi M, Brasier AR
Journal
J Extracell Vesicles
Abstract
Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and (show more...)Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation. (hide)
EV-METRIC
75% (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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bronchoalveolar lavage fluid
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
60.38 (pelleting) / 60.38 (washing)
Protein markers
EV: CD63/ HSP90/ Alix
non-EV: Grp94/ beta-actin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Bronchoalveolar lavage fluid
Origin
poly IC stimulated
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
60.38
Wash: time (min)
60
Wash: Rotor Type
TLA-100.3
Wash: speed (g)
100000
Wash: adjusted k-factor
60.38
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63,HSP90,Alix
Not detected contaminants
Grp94,beta-actin
Proteomics database
No
Characterization: Particle analysis
DLS
Report type
Median
Reported size (nm)
95
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170016 2/2 Mus musculus Cell culture supernatant dUC
Filtration
Ying, Wei 2017 55%

Study summary

Full title
All authors
Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, Ofrecio JM, Wollam J, Hernandez-Carretero A, Fu W, Li P, Olefsky JM
Journal
Cell
Abstract
MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we (show more...)MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis. (hide)
EV-METRIC
55% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
256 (pelleting) / 256 (washing)
Protein markers
EV: CD9/ CD63/ HSP70/ TSG101/ Syntenin1
non-EV: Grp94
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
adipose tissue-derived macrophages
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
Commercial EDS
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
240-360
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
256.0
Wash: time (min)
20
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
256.0
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
1.13-1.15 g/ml
Used subtypes
Yes
Characterization: Protein analysis
Protein Concentration Method
DC protein assay
Protein Concentration
9-May
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, HSP70, TSG101, Syntenin1
Not detected contaminants
Grp94
Extra information
Full UC protocol not in original article
EV170002 1/1 Homo sapiens Cell culture supernatant dUC
SEC (non-commercial)
UF
Monguió-Tortajada M 2017 50%

Study summary

Full title
All authors
Monguió-Tortajada M, Roura S, Gálvez-Montón C, Pujal JM, Aran G, Sanjurjo L, Franquesa M, Sarrias MR, Bayes-Genis A, Borràs FE
Journal
Theranostics
Abstract
Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation (show more...)Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect. (hide)
EV-METRIC
50% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + SEC (non-commercial) + UF
Adj. k-factor
138.6 (pelleting)
Protein markers
EV: CD9/ CD63/ MHC1/ MHC2/ CD73/ CD90
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Umbilical cord mesenchymal stem cells
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
138.6
Ultra filtration
Cut-off size (kDa)
100
Membrane type
NS
Size-exclusion chromatography
Total column volume (mL)
1
Sample volume/column (mL)
0.1
Resin type
Sepharose CL-2B
Characterization: Particle analysis
NTA
Report type
Mean;Median and size distribution
Reported size (nm)
160-230
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV170026 1/1 Mus musculus Cell culture supernatant DG
Filtration
dUC
Prakash Gangadaran 2017 44%

Study summary

Full title
All authors
Gangadaran P, Rajendran RL, Lee HW, Kalimuthu S, Hong CM, Jeong SY, Lee SW, Lee J, Ahn BC
Journal
J Control Release
Abstract
Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for (show more...)Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for various diseases, but their angiogenic mechanisms of therapeutic efficacy remain unclear. Here, we describe how MSC-EVs, activates VEGF receptors and downstream angiogenesis pathways. Mouse MSC-EVs were isolated from cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. In vitro migration, proliferation, and tube formation assays using endothelial cells were used to assess the angiogenic potential of MSC-EVs, and revealed higher levels of cellular migration, proliferation, and tube formation after treatment. qRT-PCR and western blotting (WB) revealed higher protein and mRNA expression of the angiogenic genes VEGFR1 and VEGFR2 in mouse SVEC-4 endothelial cells after MSC-EVs treatment. Additionally, other vital pro-angiogenic pathways (SRC, AKT, and ERK) were activated by in vitro MSC-EV treatment. WB and qRT-PCR revealed enriched presence of VEGF protein and miR-210-3p in MSC-EV. The hindlimb ischemia mouse model was established and MSC-EVs with or without Matrigel (EV-MSC+Gel) were injected into the ischemic area and blood reperfusion was monitored using molecular imaging techniques. The in vivo administration of MSC-EVs increased both blood reperfusion and the formation of new blood vessels in the ischemic limb, with the addition of matrigel enhancing this effect further by releasing EVs slowly. MSC-EVs enhance angiogenesis in ischemic limbs, most likely via the overexpression of VEGFR1 and VEGFR2 in endothelial cells. These findings reveal a novel mechanism of activating receptors by MSC-EVs influence the angiogenesis. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + Filtration + dUC
Adj. k-factor
253.9 (pelleting) / 253.9 (washing)
Protein markers
EV: Alix/ CD63
non-EV: GM130/ calnexin/ cytochromec
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
bone marrow-derived mesenchymal stem cells
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Wash: time (min)
60
Wash: Rotor Type
SW 28
Wash: speed (g)
100000
Wash: adjusted k-factor
253.9
Density cushion
Density medium
Iodixanol
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix, CD63
Not detected contaminants
GM130, calnexin, cytochrome c
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
135
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170021 1/2 Homo sapiens Cell culture supernatant DG
dUC
Liem, Michael 2017 44%

Study summary

Full title
All authors
Liem M, Ang CS, Mathivanan S
Journal
Proteomics
Abstract
Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (C (show more...)Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression. This article is protected by copyright. All rights reserved. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
253.9 (pelleting) / 89.2 (washing)
Protein markers
EV: Alix/ TSG101/ AKT/ pAKT/ beta-actin/ FAT1/ p-cadherin
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
LIM1215
EV-harvesting Medium
Serum free medium
Origin
Insulin induced
Cell viability (%)
98
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Wash: time (min)
60
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Densitometry (SYPRO Ruby)
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, TSG101,AKT,pAKT,beta-actin,FAT1,p-cadherin
Proteomics database
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-150
EV concentration
Yes
Particle yield
3.08E+07 particles/million cells
EV170021 2/2 Homo sapiens Cell culture supernatant dUC Liem, Michael 2017 44%

Study summary

Full title
All authors
Liem M, Ang CS, Mathivanan S
Journal
Proteomics
Abstract
Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (C (show more...)Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression. This article is protected by copyright. All rights reserved. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
253.9 (pelleting) / 89.2 (washing)
Protein markers
EV: Alix/ TSG101/ AKT/ beta-actin/ FAT1/ p-cadherin
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
LIM1215
EV-harvesting Medium
Serum free medium
Origin
Control condition
Cell viability (%)
98
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Wash: time (min)
60
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Densitometry (SYPRO Ruby)
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, TSG101,AKT,beta-actin,FAT1,p-cadherin
Proteomics database
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-150
EV concentration
Yes
Particle yield
1.85E+07 particles/million cells
EV170012 1/3 Homo sapiens Cell culture supernatant dUC Gualerzi, Alice 2017 44%

Study summary

Full title
All authors
Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica
Journal
Scientific Reports
Abstract
Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
158.5 (pelleting) / 158.5 (washing)
Protein markers
EV: CD9/ CD63/ Flotillin-1
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
adipose tissue mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Origin
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 55.2 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
158.5
Wash: time (min)
70
Wash: Rotor Type
Type 55.2 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
158.5
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
46.5 ± 15.8
Other particle analysis name(1)
Raman spectroscopy
EV170012 2/3 Homo sapiens Cell culture supernatant dUC Gualerzi, Alice 2017 44%

Study summary

Full title
All authors
Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica
Journal
Scientific Reports
Abstract
Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
158.5 (pelleting) / 158.5 (washing)
Protein markers
EV: CD9/ CD63/ Flotillin-1
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
dermal fibroblasts
EV-harvesting Medium
Serum free medium
Origin
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 55.2 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
158.5
Wash: time (min)
70
Wash: Rotor Type
Type 55.2 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
158.5
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
46.5 ± 15.8
Other particle analysis name(1)
Raman spectroscopy
EV170012 3/3 Homo sapiens Cell culture supernatant dUC Gualerzi, Alice 2017 44%

Study summary

Full title
All authors
Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica
Journal
Scientific Reports
Abstract
Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
158.5 (pelleting) / 158.5 (washing)
Protein markers
EV: CD9/ CD63/ Flotillin-1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
bone marrow-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Origin
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 55.2 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
158.5
Wash: time (min)
70
Wash: Rotor Type
Type 55.2 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
158.5
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
46.5 ± 15.8
Other particle analysis name(1)
Raman spectroscopy
EV170003 1/1 Mus musculus Cell culture supernatant dUC Nager AR 2017 44%

Study summary

Full title
All authors
Nager AR, Goldstein JS, Herranz-Pérez V, Portran D, Ye F, Garcia-Verdugo JM, Nachury MV
Journal
Cell
Abstract
Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. H (show more...)Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Ciliary Ectosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
253.9 (pelleting) / 99.86 (washing)
Protein markers
EV: CD81/ Arl13B/ BiotinylatedGPCRs
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
mIMCD3
EV-harvesting Medium
Serum-containing medium
Origin
GPCR signaling in BBS mutant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Wash: time (min)
90
Wash: Rotor Type
TLS-55
Wash: speed (g)
100000
Wash: adjusted k-factor
99.86
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Detected EV-associated proteins
CD81, Arl13B
Characterization: Particle analysis
EM
EM-type
Transmission-EM/ Immune-EM
Image type
Close-up, Wide-field
Report size (nm)
70-120
EV170006 1/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD147/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170006 2/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD147/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Inguinal hernia
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170006 3/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD147/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Colon carcinoma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170006 4/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD147/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Hepatocellular carcinoma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170006 5/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD147/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Liver tumour
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170006 6/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD147/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Cirrhosis
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170006 7/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Cholangiocarcinoma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170006 8/8 Homo sapiens Serum dUC Julich-Haertel H 2017 37%

Study summary

Full title
All authors
Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M
Journal
J Hepatol
Abstract
BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide)
EV-METRIC
37% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
microparticle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
284.4 (pelleting)
Protein markers
EV: ANXA5/ EpCAM/ CD133/ ASGPR1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Origin
Non small cell lung carcinoma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
284.4
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry
Type of Flow cytometry
MACSQuant® Analyzer 10
Calibration bead size
0.2,0.5
Extra information
We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).
EV170024 1/3 Homo sapiens Cell culture supernatant dUC
Filtration
Volgers C 2017 33%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Savelkoul PHM, Stassen FRM
Journal
Abstract
During infection, the release of nano-sized membrane vesicle is a process which is common both for b (show more...)During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: CD63/ CD81
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
THP1
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63,CD81
Flow cytometry specific beads
Selected surface protein(s)
CD63
EV170024 2/3 Moraxella catarrhalis Cell culture supernatant dUC
Filtration
Volgers C 2017 33%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Savelkoul PHM, Stassen FRM
Journal
Abstract
During infection, the release of nano-sized membrane vesicle is a process which is common both for b (show more...)During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: Moraxella catarrhalis antigen
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Moraxella catarrhalis
Sample Type
Cell culture supernatant
EV-producing cells
Moraxella catarrhalis
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Moraxella catarrhalis antigen
Flow cytometry specific beads
Selected surface protein(s)
Moraxella catarrhalis antigen
Characterization: Particle analysis
TRPS
EV concentration
Yes
Particle yield
1.50E+09 particles/ml start sample
EV170024 3/3 Pseudomonas aeruginosa Cell culture supernatant dUC
Filtration
Volgers C 2017 33%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Savelkoul PHM, Stassen FRM
Journal
Abstract
During infection, the release of nano-sized membrane vesicle is a process which is common both for b (show more...)During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: Pseudomonas aeruginosa antigen
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Pseudomonas aeruginosa
Sample Type
Cell culture supernatant
EV-producing cells
Pseudomonas aeruginosa
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Pseudomonas aeruginosa antigen
Flow cytometry specific beads
Selected surface protein(s)
Pseudomonas aeruginosa antigen
Characterization: Particle analysis
TRPS
EV concentration
Yes
Particle yield
1.00E+09 particles/ml start sample
EV170000 1/2 Mus musculus Serum dUC
Filtration
Thomou T 2017 29%

Study summary

Full title
All authors
Thomou T, Mori MA, Dreyfuss JM, Konishi M, Sakaguchi M, Wolfrum C, Rao TN, Winnay JN, Garcia-Martin R, Grinspoon SK, Gorden P, Kahn
Journal
Nature
Abstract
Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism thro (show more...)Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine. (hide)
EV-METRIC
29% (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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Protein markers
EV: CD63/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Serum
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: speed (g)
100000
Wash: time (min)
60
Wash: speed (g)
100000
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
EM
EM-type
Transmission-EM/ Immune-EM
Image type
Close-up, Wide-field
Report size (nm)
80-200
Other particle analysis name(1)
EXOCET ELISA assay
EV-concentration
Yes
EV170005 1/4 Homo sapiens Cell culture supernatant dUC
SEC
UF
Suárez H 2017 28%

Study summary

Full title
All authors
Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M
Journal
Sci Rep
Abstract
Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide)
EV-METRIC
28% (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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + SEC + UF
Protein markers
EV: CD9/ CD63/ CD81/ MHC1/ CD59
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
SK-MEL103
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
Not spec
Membrane type
Not specified
Size-exclusion chromatography
Total column volume (mL)
20
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
148.16
Characterization: Particle analysis
NTA
Report type
Median
EV170005 4/4 Homo sapiens Cell culture supernatant dUC
SEC
UF
Suárez H 2017 28%

Study summary

Full title
All authors
Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M
Journal
Sci Rep
Abstract
Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide)
EV-METRIC
28% (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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + SEC + UF
Protein markers
EV: CD9/ CD63/ CD81/ MHC1/ CD59
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary T-lymphoblasts
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
Not spec
Membrane type
Not specified
Size-exclusion chromatography
Total column volume (mL)
20
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Concentration
116.85
Characterization: Particle analysis
NTA
Report type
Median
EV170005 3/4 Homo sapiens Cell culture supernatant dUC Suárez H 2017 25%

Study summary

Full title
All authors
Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M
Journal
Sci Rep
Abstract
Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide)
EV-METRIC
25% (64th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
264.9 (pelleting) / 264.9 (washing)
Protein markers
EV: CD9/ CD59
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
SK-MEL103
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
AH-627
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
264.9
Wash: time (min)
120
Wash: Rotor Type
AH-627
Wash: speed (g)
100000
Wash: adjusted k-factor
264.9
Characterization: Protein analysis
Protein Concentration Method
BCA
Characterization: Particle analysis
NTA
Report type
Median
EV170035 4/4 Homo sapiens Cell culture supernatant Commercial method Vestad B 2017 16%

Study summary

Full title
All authors
Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Øvstebø R
Journal
J Extracell Vesicles
Abstract
Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in (show more...)Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted. (hide)
EV-METRIC
16% (48th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method
Protein markers
EV: CD81
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Jurkat E61
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Origin
Control condition
Isolation Method
Commercial kit
Total Exosome Isolation
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Mean,Mode
Reported size (nm)
Mean: 130-144;Mode: 91-99
EV concentration
Yes
Particle yield
5.1E8-5.6E8
EM
EM-type
Immune-EM
Image type
Wide-field
Extra information
This was a technical report and a variation study focusing on NTA, detailed characterization of EVs less important. NTA data of these EVs not obtained using instrument-optimized settings.
EV170035 1/4 Neisseria meningitidis Cell culture supernatant dUC Vestad B 2017 14%

Study summary

Full title
All authors
Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Øvstebø R
Journal
J Extracell Vesicles
Abstract
Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in (show more...)Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Outer membrane vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Neisseria meningitidis
Sample Type
Cell culture supernatant
EV-producing cells
Neisseria meningitidis
EV-harvesting Medium
Not specified
Origin
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: speed (g)
140000
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Mean,Mode
Reported size (nm)
Mean: 138-156;Mode: 102-122
EV concentration
Yes
Particle yield
5.8E8-8.9E8
EM
EM-type
Transmission-EM
Image type
Wide-field
Extra information
This was a technical report and a variation study focusing on NTA, detailed characterization of EVs less important. NTA data of these EVs not obtained using instrument-optimized settings.
EV170035 2/4 Homo sapiens Cell culture supernatant dUC Vestad B 2017 14%

Study summary

Full title
All authors
Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Øvstebø R
Journal
J Extracell Vesicles
Abstract
Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in (show more...)Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
(shedding) microvesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary monocytes
EV-harvesting Medium
EV-depleted serum
Origin
Stimulated with E. coli LPS
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Pelleting: speed (g)
17000
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Mean,Mode
Reported size (nm)
Mean: 167-171;Mode: 119-127
EV concentration
Yes
Particle yield
3.9E8-4.3E8
EM
EM-type
Transmission-EM
Image type
Wide-field
Extra information
This was a technical report and a variation study focusing on NTA, detailed characterization of EVs less important. NTA data of these EVs obtained using instrument-optimized settings.
EV170035 3/4 Homo sapiens Cell culture supernatant dUC Vestad B 2017 14%

Study summary

Full title
All authors
Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Øvstebø R
Journal
J Extracell Vesicles
Abstract
Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in (show more...)Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PC3
EV-harvesting Medium
Serum free medium
Origin
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: speed (g)
100000
Wash: time (min)
70
Wash: speed (g)
100000
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Mean,Mode
Reported size (nm)
Mean:125-135;Mode:89-92
EV concentration
Yes
Particle yield
2.9E8-3.1E8
EM
EM-type
Immune-EM
Image type
Wide-field
Extra information
This was a technical report and a variation study focusing on NTA, detailed characterization of EVs less important. NTA data of these EVs obtained using instrument-optimized settings.
EV170022 1/5 Streptococcus pneumoniae Cell culture supernatant dUC
Filtration
SEC
UF
Volgers C 2017 14%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM
Journal
FEMS Microbiol Lett
Abstract
Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: Streptococcus pneumoniae antigen
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biogenesis/cargo sorting
Sample
Species
Streptococcus pneumoniae
Sample Type
Cell culture supernatant
EV-producing cells
Streptococcus pneumoniae
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry specific beads
Selected surface protein(s)
Streptococcus pneumoniae antigen
Characterization: Particle analysis
TRPS
EV concentration
Yes
EV170022 2/5 Moraxella catarrhalis Cell culture supernatant dUC
Filtration
SEC
UF
Volgers C 2017 14%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM
Journal
FEMS Microbiol Lett
Abstract
Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: Moraxella catarrhalis antigen
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biogenesis/cargo sorting
Sample
Species
Moraxella catarrhalis
Sample Type
Cell culture supernatant
EV-producing cells
Moraxella catarrhalis
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry specific beads
Selected surface protein(s)
Moraxella catarrhalis antigen
Characterization: Particle analysis
TRPS
EV concentration
Yes
EV170022 3/5 Haemophilus influenzae Cell culture supernatant dUC
Filtration
SEC
UF
Volgers C 2017 14%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM
Journal
FEMS Microbiol Lett
Abstract
Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: non-typeable Haemophilus influenzae antigen
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biogenesis/cargo sorting
Sample
Species
Haemophilus influenzae
Sample Type
Cell culture supernatant
EV-producing cells
non-typeable Haemophilus influenzae
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry specific beads
Selected surface protein(s)
non-typeable Haemophilus influenzae antigen
Characterization: Particle analysis
TRPS
EV concentration
Yes
EV170022 4/5 Homo sapiens Cell culture supernatant dUC
Filtration
SEC
UF
Volgers C 2017 14%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM
Journal
FEMS Microbiol Lett
Abstract
Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: CD63/ CD81
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
THP1
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry specific beads
Selected surface protein(s)
CD63
Characterization: Particle analysis
TRPS
EV concentration
Yes
EV170022 5/5 Pseudomonas aeruginosa Cell culture supernatant dUC
Filtration
SEC
UF
Volgers C 2017 14%

Study summary

Full title
All authors
Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM
Journal
FEMS Microbiol Lett
Abstract
Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: Pseudomonas aeruginosa antigen
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biogenesis/cargo sorting
Sample
Species
Pseudomonas aeruginosa
Sample Type
Cell culture supernatant
EV-producing cells
Pseudomonas aeruginosa
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Flow cytometry specific beads
Selected surface protein(s)
Pseudomonas aeruginosa antigen
Characterization: Particle analysis
TRPS
EV concentration
Yes
EV170023 1/5 Haemophilus influenzae Cell culture supernatant dUC
Filtration
SEC
UF
Volgers C 2017 14%

Study summary

Full title
All authors
Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM
Journal
Scientific Reports
Abstract
Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
membrane vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + UF
Protein markers
EV: non-typeable Haemophilus influenzae antigen
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Haemophilus influenzae
Sample Type
Cell culture supernatant
EV-producing cells
non-typeable Haemophilus influenzae
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis