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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Experiment number
  • Experiments differ in isolation protocol
Experiment number
  • Experiments differ in isolation protocol
Experiment number
  • Experiments differ in isolation protocol
Experiment number
  • Experiments differ in Vesicle type
Experiment number
  • Experiments differ in Vesicle type
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample condition
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  • Experiments differ in Sample condition
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  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV170028 1/1 Homo sapiens Synovial fluid DG
(d)(U)C
SEC
Foers AD 2017 100%

Study summary

Full title
All authors
Foers AD, Chatfield S, Dagley LF, Scicluna BJ, Webb AI, Cheng L, Hill AF, Wicks IP, Pang KC.
Journal
J Extracell Vesicles
Abstract
As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesic (show more...)As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease. (hide)
EV-METRIC
100% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Synovial fluid
Sample origin
Arthritis
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
SEC
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: TSG101/ Rab27b/ Annexin1/ Syntenin/ HSP70/ Flotillin-1
non-EV: Fibronectin/ Albumin/ ApoA1
Proteomics
yes
Show all info
Study aim
New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Synovial fluid
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
90
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0.25
Highest density fraction
2
Sample volume (mL)
0.65
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 40 Ti
Speed (g)
202000
Duration (min)
1100
Fraction volume (mL)
1.05
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: duration (min)
90
Pelleting: rotor type
Type 70.1Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
122.2
Size-exclusion chromatography
Total column volume (mL)
320
Sample volume/column (mL)
13
Resin type
Sephacryl S-500 HR
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin-1, HSP70, TSG101, Syntenin, Rab27b, Annexin1
Not detected contaminants
Albumin, ApoA1
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
x
EV concentration
Yes
Particle yield
x
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170008 1/3 Homo sapiens primary monocyte derived dendritic cell DG
(d)(U)C
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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: CD63/ MHC2/ CD9/ CD9,MHC2
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
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
Yes
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
Protein Concentration Method
microBCA
Protein Yield (µg)
2.9+-0.3
Western Blot
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: Lipid analysis
No
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
EM protein
MHC2
Image type
Close-up, Wide-field
EV170008 2/3 Homo sapiens primary monocyte derived dendritic cell DG
(d)(U)C
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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
2097 (pelleting) / 2097 (washing)
Protein markers
EV: CD63/ MHC2/ CD9/ CD9,MHC2
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
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
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
Protein Concentration Method
microBCA
Protein Yield (µg)
1.7+-0.3
Western Blot
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: Lipid analysis
No
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
EM protein
MHC2
Image type
Close-up, Wide-field
EV170008 3/3 Homo sapiens primary monocyte derived dendritic cell DG
(d)(U)C
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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
209.7 (pelleting) / 209.7 (washing)
Protein markers
EV: CD63/ MHC2/ CD9/ CD9,MHC2
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
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
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
Protein Concentration Method
microBCA
Protein Yield (µg)
1.1+-0.2
Western Blot
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: Lipid analysis
No
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
EM protein
MHC2
Image type
Close-up, Wide-field
EV170001 1/1 Homo sapiens MCF7 Rab27b-GFP DG
(d)(U)C
Filtration
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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
UF
Protein markers
EV: Alix/ CD81/ TSG101
non-EV: PMP70/ Argonaute-2
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
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
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
Detected EV-associated proteins
Alix/ CD81/ TSG101
Not detected contaminants
Argonaute-2/ PMP70
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
113
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170046 1/2 Sus scrofa Primary retinal pigmented epithelium cells DG
(d)(U)C
UF
Klingeborn M 2017 88%

Study summary

Full title
All authors
Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C
Journal
Sci Rep
Abstract
The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Basolateral side of polarized layer
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
UF
Adj. k-factor
253.9 (pelleting) / 253.9 (washing)
Protein markers
EV: TSG101/ Syntenin-1
non-EV: Calreticulin
Proteomics
yes
Show all info
Study aim
New methodological development, Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
EV-producing cells
Primary retinal pigmented epithelium cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
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
SW 28
Wash: speed (g)
100000
Wash: adjusted k-factor
253.9
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
1
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Characterization: Protein analysis
Protein Concentration Method
Pierce 660nm Protein Assay
Western Blot
Detected EV-associated proteins
TSG101, Syntenin-1
Not detected contaminants
Calreticulin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
132.2±13.1
EM
EM-type
Transmission-EM
Image type
Wide-field
EV170046 2/2 Sus scrofa Primary retinal pigmented epithelium cells DG
(d)(U)C
UF
Klingeborn M 2017 88%

Study summary

Full title
All authors
Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C
Journal
Sci Rep
Abstract
The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Apical side of polarized layer
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
UF
Adj. k-factor
253.9 (pelleting) / 253.9 (washing)
Protein markers
EV: TSG101/ Syntenin-1
non-EV: Calreticulin
Proteomics
yes
Show all info
Study aim
New methodological development, Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
EV-producing cells
Primary retinal pigmented epithelium cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
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
SW 28
Wash: speed (g)
100000
Wash: adjusted k-factor
253.9
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
1
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Characterization: Protein analysis
Protein Concentration Method
Pierce 660nm Protein Assay
Western Blot
Detected EV-associated proteins
TSG101, Syntenin-1
Not detected contaminants
Calreticulin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
118.6±9.9
EM
EM-type
Transmission-EM
Image type
Close-up
EV170016 1/2 Mus musculus adipose tissue-derived macrophages DG
(d)(U)C
Filtration
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% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Obesitas
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Adj. k-factor
256 (pelleting) / 256 (washing)
Protein markers
EV: TSG101/ HSP70/ Syntenin1/ CD63/ CD9
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
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
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 Yield (µg)
9-May
Western Blot
Detected EV-associated proteins
CD9, CD63, HSP70, TSG101, Syntenin1
Not detected contaminants
Grp94
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
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
(d)(U)C
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% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Adipose tissue
Sample origin
breast cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Adj. k-factor
138.6 (pelleting) / 138.6 (washing)
Protein markers
EV: CD63/ HSP70/ FABP4/ Flotillin-1/ CD9
non-EV: Prohibitin/ GM130/ Calreticulin
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Adipose tissue
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
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
Detected EV-associated proteins
CD9, Flotillin-1, HSP70, FABP4
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
116
EV concentration
Yes
Particle yield
2.2 10E09
EM
EM-type
Immune-EM
EM protein
CD63
Image type
Close-up, Wide-field
Report size (nm)
20-200
EV210202 1/7 Mus musculus 3T3-L1 DG
(d)(U)C
Durcin, Maëva 2017 78%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Large extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.14-1.24
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
3T3-L1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
13000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
13000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
31
Lowest density fraction
0.4M
Highest density fraction
2M
Total gradient volume, incl. sample (mL)
12000
Sample volume (mL)
500
Orientation
Top-down
Rotor type
Not specified
Speed (g)
200000
Duration (min)
1080
Fraction volume (mL)
1000
Fraction processing
Centrifugation
Pelleting: volume per fraction
6000
Pelleting: duration (min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Mfge8/ caveolin-1/ b-actin/ Flotillin2/ TSG101/ Alix
Not detected EV-associated proteins
CD81
Flow cytometry
Type of Flow cytometry
MACSQuant flow cytometer
Calibration bead size
4µm
Detected EV-associated proteins
Annexin V
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
Particle yield
EV secreted per adipocyte;Yes, other: 11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-100
EV210202 2/7 Mus musculus 3T3-L1 DG
(d)(U)C
Durcin, Maëva 2017 78%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.14-1.20
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
3T3-L1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
31
Lowest density fraction
0.4M
Highest density fraction
2M
Total gradient volume, incl. sample (mL)
12000
Sample volume (mL)
500
Orientation
Top-down
Rotor type
Not specified
Speed (g)
200000
Duration (min)
1080
Fraction volume (mL)
1000
Fraction processing
Centrifugation
Pelleting: volume per fraction
6000
Pelleting: duration (min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Mfge8/ Flotillin2/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
caveolin-1/ b-actin
Flow cytometry
Type of Flow cytometry
MACSQuant flow cytometer
Calibration bead size
4µm
Not detected EV-associated proteins
Annexin V
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
100
EV concentration
Yes
Particle yield
EV secreted per adipocyte;Yes, other: 843
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
90-800
EV170067 1/6 Homo sapiens HMC-1 DG
(d)(U)C
Lässer C 2017 78%

Study summary

Full title
All authors
Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J.
Journal
RNA Biol
Abstract
Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Extracellular vesicle-like structures
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Flotillin1/ ANXA2/ CD9
non-EV:
Proteomics
yes
EV density (g/ml)
1.09 - 1.31
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HMC-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
16
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
35
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
175000
Duration (min)
960
Fraction volume (mL)
3.9
Fraction processing
Centrifugation
Pelleting: duration (min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.09-1.21 g/mL
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ ANXA2/ TSG101/ CD81
Flow cytometry specific beads
Detected EV-associated proteins
CD9/ CD63/ CD81
Proteomics database
Yes
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing;Microarray
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Immuno-EM
EM protein
CD63
Image type
Close-up, Wide-field
Report size (nm)
92
EV170064 1/2 Bos taurus Milk DG
(d)(U)C
Samuel, M 2017 78%

Study summary

Full title
All authors
Samuel M, Chisanga D, Liem M, Keerthikumar S, Anand S, Ang CS, Adda CG, Versteegen E, Jois M, Mathivanan S
Journal
Abstract
Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. Th (show more...)Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth. (hide)
EV-METRIC
78% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Milk
Sample origin
Control condition
Focus vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: Alix/ TSG101
non-EV: Albumin/ GM130
Proteomics
yes
EV density (g/ml)
1.16
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Bos taurus
Sample Type
Milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
1080
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
0.5
Wash: time (min)
60
Wash: Rotor Type
SW 28
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
TSG101
Not detected EV-associated proteins
Alix
Detected contaminants
Albumin
Not detected contaminants
GM130
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
145
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170064 2/2 Bos taurus Milk DG
(d)(U)C
Samuel, M 2017 78%

Study summary

Full title
All authors
Samuel M, Chisanga D, Liem M, Keerthikumar S, Anand S, Ang CS, Adda CG, Versteegen E, Jois M, Mathivanan S
Journal
Abstract
Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. Th (show more...)Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth. (hide)
EV-METRIC
78% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Milk
Sample origin
Colostrum
Focus vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: / Alix/ TSG101
non-EV: Albumin/ GM130
Proteomics
yes
EV density (g/ml)
1.16
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Bos taurus
Sample Type
Milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
1080
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
0.5
Wash: time (min)
60
Wash: Rotor Type
SW 28
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ TSG101
Not detected EV-associated proteins
Detected contaminants
Albumin
Not detected contaminants
GM130
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
125
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170065 1/1 Homo sapiens Primary tumor-derived fibroblasts (d)(U)C Bhome R 2017 77%

Study summary

Full title
All authors
Bhome R, Goh RW1, Bullock MD, Pillar N, Thirdborough SM, Mellone M, Mirnezami R, Galea D, Veselkov K, Gu Q, Underwood TJ, Primrose JN, De Wever O, Shomron N, Sayan AE, Mirnezami AH.
Journal
Aging (Albany NY)
Abstract
Colorectal cancer is a global disease with increasing incidence. Mortality is largely attributed to (show more...)Colorectal cancer is a global disease with increasing incidence. Mortality is largely attributed to metastatic spread and therefore, a mechanistic dissection of the signals which influence tumor progression is needed. Cancer stroma plays a critical role in tumor proliferation, invasion and chemoresistance. Here, we sought to identify and characterize exosomal microRNAs as mediators of stromal-tumor signaling. In vitro, we demonstrated that fibroblast exosomes are transferred to colorectal cancer cells, with a resultant increase in cellular microRNA levels, impacting proliferation and chemoresistance. To probe this further, exosomal microRNAs were profiled from paired patient-derived normal and cancer-associated fibroblasts, from an ongoing prospective biomarker study. An exosomal cancer-associated fibroblast signature consisting of microRNAs 329, 181a, 199b, 382, 215 and 21 was identified. Of these, miR-21 had highest abundance and was enriched in exosomes. Orthotopic xenografts established with miR-21-overexpressing fibroblasts and CRC cells led to increased liver metastases compared to those established with control fibroblasts. Our data provide a novel stromal exosome signature in colorectal cancer, which has potential for biomarker validation. Furthermore, we confirmed the importance of stromal miR-21 in colorectal cancer progression using an orthotopic model, and propose that exosomes are a vehicle for miR-21 transfer between stromal fibroblasts and cancer cells. (hide)
EV-METRIC
77% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Colorectal cancer
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
88.25 (pelleting) / 88.25 (washing)
Protein markers
EV: Alix/ CD81/ TSG101/ CD63
non-EV: cytochrome c/ GM130/ cytochromec
Proteomics
no
Show all info
Study aim
Function, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary tumor-derived fibroblasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 50.3 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
88.25
Wash: time (min)
70
Wash: Rotor Type
Type 50.3 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
88.25
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
100
Western Blot
Detected EV-associated proteins
Alix, CD63, CD81, TSG101
Not detected contaminants
GM130, cytochrome c
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
113
EV concentration
Yes
Particle yield
1570000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170063 1/2 Homo sapiens Jurkat DG
(d)(U)C
Filtration
Nemeth, Andrea 2017 75%

Study summary

Full title
All authors
Németh A, Orgovan N, Sódar BW, Osteikoetxea X, Pálóczi K, Szabó-Taylor KÉ, Vukman KV, Kittel Á, Turiák L, Wiener Z, Tóth S, Drahos L, Vékey K, Horvath R, Buzás EI.
Journal
Sci Rep
Abstract
Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesi (show more...)Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes. (hide)
EV-METRIC
75% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Cyprofloxacin
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Adj. k-factor
115.3 (pelleting) / 115.3 (washing)
Protein markers
EV: CD63/ HistoneH2B
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Jurkat
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
115.3
Wash: time (min)
70
Wash: Rotor Type
MLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
115.3
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: duration (min)
180
Pelleting: speed (g)
100000
Filtration steps
> 0.45 µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV170063 2/2 Homo sapiens Jurkat DG
(d)(U)C
Filtration
Nemeth, Andrea 2017 75%

Study summary

Full title
All authors
Németh A, Orgovan N, Sódar BW, Osteikoetxea X, Pálóczi K, Szabó-Taylor KÉ, Vukman KV, Kittel Á, Turiák L, Wiener Z, Tóth S, Drahos L, Vékey K, Horvath R, Buzás EI.
Journal
Sci Rep
Abstract
Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesi (show more...)Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes. (hide)
EV-METRIC
75% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Adj. k-factor
115.3 (pelleting) / 115.3 (washing)
Protein markers
EV: CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Jurkat
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
115.3
Wash: time (min)
70
Wash: Rotor Type
MLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
115.3
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: duration (min)
180
Pelleting: speed (g)
100000
Filtration steps
> 0.45 µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV170017 1/1 Mus musculus Bronchoalveolar lavage fluid (d)(U)C 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% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bronchoalveolar lavage fluid
Sample origin
poly IC stimulated
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
60.38 (pelleting) / 60.38 (washing)
Protein markers
EV: Alix/ CD63,HSP90,Alix/ HSP90/ CD63
non-EV: Grp94,beta-actin/ Grp94/ beta-actin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Bronchoalveolar lavage fluid
Separation Method
(Differential) (ultra)centrifugation
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 performed
Yes
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
Detected EV-associated proteins
CD63,HSP90,Alix
Not detected contaminants
Grp94,beta-actin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Median
Reported size (nm)
95
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230813 2/6 Escherichia coli W3110 (d)(U)C
DG
Filtration
UF
Kim OY 2017 72%

Study summary

Full title
All authors
Kim OY, Park HT, Dinh NTH, Choi SJ, Lee J, Kim JH, Lee SW, Gho YS
Journal
Nat Commun
Abstract
Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteoli (show more...)Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects.Bacterial outer membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response. (hide)
EV-METRIC
72% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
msbB deletion mutant
Focus vesicles
outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Ultrafiltration
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
W3110
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: speed (g)
150000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Orientation
Bottom-up
Speed (g)
200000
Duration (min)
120
Fraction processing
None
Filtration steps
0.2 or 0.22 µm/ 0.45 µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Hollowfiber
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
ProteomeXchange Consortium
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
38.7
EM
EM-type
Transmission-EM
Image type
Wide-field
EV230753 1/1 Aggregatibacter actinomycetemcomitans 173 (d)(U)C
DG
Filtration
Kieselbach T 2017 72%

Study summary

Full title
All authors
Kieselbach T, Oscarsson J
Journal
Data Brief
Abstract
The Gram-negative bacterium is an oral and systemic pathogen, which is linked to aggressive forms o (show more...)The Gram-negative bacterium is an oral and systemic pathogen, which is linked to aggressive forms of periodontitis and can be associated with endocarditis. The outer membrane vesicles (OMVs) of this species contain effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA), which they can deliver into human host cells. The OMVs can also activate innate immunity through NOD1- and NOD2-active pathogen-associated molecular patterns. This dataset provides a proteome of highly purified OMVs from serotype strain 173. The experimental data do not only include the raw data of the LC-MS/MS analysis of four independent preparations of purified OMVs but also the mass lists of the processed data and the Mascot.dat files from the database searches. In total 501 proteins are identified, of which 151 are detected in at least three of four independent preparations. In addition, this dataset contains the COG definitions and the predicted subcellular locations (PSORTb 3.0) for the entire genome of serotype strain SC1083, which is used for the evaluation of the LC-MS/MS data. These data are deposited in ProteomeXchange in the public dataset PXD002509. In addition, a scientific interpretation of this dataset by Kieselbach et al. (2015) [2] is available at http://dx.doi.org/10.1371/journal.pone.0138591. (hide)
EV-METRIC
72% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
outer membrane vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
15195 (pelleting) / 0 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Aggregatibacter actinomycetemcomitans
Sample Type
Cell culture supernatant
EV-producing cells
173
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
85 000
Pelleting: adjusted k-factor
15195
Wash: volume per pellet (ml)
not specified
Wash: time (min)
120
Wash: speed (g)
85000
Wash: adjusted k-factor
TDB
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
7
Lowest density fraction
10%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
4,17
Sample volume (mL)
0,15
Orientation
Bottom-up
Speed (g)
180000
Duration (min)
180
Fraction volume (mL)
0,2
Fraction processing
Centrifugation
Pelleting: volume per fraction
not spec
Pelleting: speed (g)
16000
Pelleting: adjusted k-factor
95526
Pelleting-wash: volume per pellet (mL)
not spec
Pelleting-wash: speed (g)
JA-18.1
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
ProteomeXchange
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
EV230812 2/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 71%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
71% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
ΔlnyI
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
14
Sample volume (mL)
1
Orientation
Bottom-up
Speed (g)
100400
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4mL
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
1.857
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV concentration
Yes
EV230812 3/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 71%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
71% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
ΔlnyI + lnyI (complement)
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
14
Sample volume (mL)
1
Orientation
Bottom-up
Speed (g)
100400
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4mL
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
1.857
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV concentration
Yes
EV230783 1/1 Salmonella enterica chi 3744 (d)(U)C
DG
Filtration
Liu Q 2017 67%

Study summary

Full title
All authors
Liu Q, Yi J, Liang K, Zhang X, Liu Q
Journal
J Microbiol Biotechnol
Abstract
Foodborne contamination and salmonellosis caused by Enteritidis (. Enteritidis) are a significant t (show more...)Foodborne contamination and salmonellosis caused by Enteritidis (. Enteritidis) are a significant threat to human health and poultry enterprises. Outer membrane vesicles (OMVs), which are naturally secreted by gram-negative bacteria, could be a good vaccine option because they have many biologically active substances, including lipopolysaccharides (LPS), outer membrane proteins (OMPs), and phospholipids, as well as periplasmic components. In the present study, we purified OMVs derived from . Enteritidis and analyzed their characteristics through silver staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis. In total, 108 proteins were identified in . Enteritidis OMVs through liquid chromatography tandem mass spectrometry analysis, and OMPs, periplasmic proteins, and extracellular proteins (49.9% of total proteins) were found to be enriched in the OMVs compared with bacterial cells. Furthermore, native OMVs used in immunizations by either the intranasal route or the intraperitoneal route could elicit significant humoral and mucosal immune responses and provide strong protective efficiency against a lethal dose (~100-fold LD) of the wild-type . Enteritidis infection. These results indicated that . Enteritidis OMVs might be an ideal vaccine strategy for preventing . Enteritidis diseases. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
outer membrane vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: SopD/ TufA/ SEN0876
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Salmonella enterica
Sample Type
Cell culture supernatant
EV-producing cells
chi 3744
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: speed (g)
40000
Density gradient
Type
Discontinuous
Lowest density fraction
20%
Highest density fraction
45%
Speed (g)
150000
Duration (min)
overnight
Fraction processing
Centrifugation
Pelleting: speed (g)
40000
Filtration steps
Between 0.22 and 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Proteomics database
No
Detected contaminants
SopD/ TufA/ SEN0876
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV170067 2/6 Homo sapiens HMC-1 DG
(d)(U)C
Filtration
Lässer C 2017 67%

Study summary

Full title
All authors
Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J.
Journal
RNA Biol
Abstract
Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Extracellular vesicle-like structures
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ Flotillin1/ ANXA2/ CD9
non-EV:
Proteomics
yes
EV density (g/ml)
1.09-1.31
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HMC-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
16
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
35
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
175000
Duration (min)
960
Fraction volume (mL)
3.9
Fraction processing
Centrifugation
Pelleting: duration (min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.24-1.31 g/mL
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ TSG101/ CD81
Not detected EV-associated proteins
ANXA2
Flow cytometry specific beads
Detected EV-associated proteins
CD9/ CD63/ CD81
Proteomics database
Yes
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing;Microarray
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Immuno-EM
EM protein
CD63
Image type
Wide-field
Report size (nm)
51
EV220172 1/2 Homo sapiens Blood plasma SEC (non-commercial)
UF
Endzeliņš E 2017 63%

Study summary

Full title
All authors
Endzeliņš E, Berger A, Melne V, Bajo-Santos C, Soboļevska K, Ābols A, Rodriguez M, Šantare D, Rudņickiha A, Lietuvietis V, Llorente A, Linē A
Journal
BMC Cancer
Abstract
Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detec (show more...)Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. (hide)
EV-METRIC
63% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Benign prostatic hyperplasia
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Size-exclusion chromatography (non-commercial)
Ultrafiltration
Protein markers
EV: CD9/ TSG101/ Actin beta
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Ultra filtration
Cut-off size (kDa)
3 Kda
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.4
Resin type
IsoaddSECResinType
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ Actin beta
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
1000
RNAse treatment
Yes
RNAse type
RNase A
RNAse concentration
10
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-150
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
25-60
EV220172 2/2 Homo sapiens Blood plasma SEC (non-commercial)
UF
Endzeliņš E 2017 63%

Study summary

Full title
All authors
Endzeliņš E, Berger A, Melne V, Bajo-Santos C, Soboļevska K, Ābols A, Rodriguez M, Šantare D, Rudņickiha A, Lietuvietis V, Llorente A, Linē A
Journal
BMC Cancer
Abstract
Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detec (show more...)Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. (hide)
EV-METRIC
63% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Prostate cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Size-exclusion chromatography (non-commercial)
Ultrafiltration
Protein markers
EV: CD9/ TSG101/ Actin beta
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Ultra filtration
Cut-off size (kDa)
3 Kda
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.4
Resin type
IsoaddSECResinType
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ Actin beta
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
1000
RNAse treatment
Yes
RNAse type
RNase A
RNAse concentration
10
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-150
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
25-60
EV170061 1/3 Homo sapiens BEAS2B (d)(U)C
Filtration
SEC
UF
Benedikter BJ 2017 62%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, Wouters EFM, Savelkoul PH, Lopez-Iglesias C, Koenen RR, Rohde GGU, Stassen FRM
Journal
J Cell Sci
Abstract
Appropriate isolation methods are essential for unravelling the relative contribution of extracellul (show more...)Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. (hide)
EV-METRIC
62% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC
UF
Protein markers
EV: CD81/ CD63/ CD9/ MFGE8
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
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
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD63, CD81, MFGE8
Flow cytometry specific beads
Selected surface protein(s)
CD9, CD63, CD81
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Median
Reported size (nm)
61.9
EV concentration
Yes
Particle yield
3.00E+01
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
25-200
EV170054 5/5 Homo sapiens Urine (d)(U)C
Filtration
SEC
Gheinani AH 2017 62%

Study summary

Full title
All authors
Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, Monastyrskaya K
Journal
Sci Rep
Abstract
Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RN (show more...)Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50-150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies. (hide)
EV-METRIC
62% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC
Protein markers
EV: TSG101/ CD81/ TSG101,CD81,CD9/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Separation Method
(Differential) (ultra)centrifugation
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 performed
No
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
23
Sample volume/column (mL)
0.4
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Protein Yield (µg)
6.8
Western Blot
Detected EV-associated proteins
TSG101,CD81,CD9
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mode;mean;size range/distribution;D10;D50;D90
Reported size (nm)
106
EV concentration
Yes
Particle yield
see Fig1A
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Extra information
In the optimized protocol, the 16000g pellet was treated with DTT, pelleted and supernatant was pooled with supernatant from the 16000g step to increase yield (see figure 4)
EV230813 4/6 Staphylococcus aureus S. aureus (d)(U)C
DG
Filtration
UF
Kim OY 2017 58%

Study summary

Full title
All authors
Kim OY, Park HT, Dinh NTH, Choi SJ, Lee J, Kim JH, Lee SW, Gho YS
Journal
Nat Commun
Abstract
Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteoli (show more...)Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects.Bacterial outer membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response. (hide)
EV-METRIC
58% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Ultrafiltration
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Staphylococcus aureus
Sample Type
Cell culture supernatant
EV-producing cells
S. aureus
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: speed (g)
150000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Orientation
Bottom-up
Speed (g)
200000
Duration (min)
120
Fraction processing
None
Filtration steps
0.2 or 0.22 µm/ 0.45 µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Hollowfiber
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
ProteomeXchange Consortium
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
EV230812 1/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 57%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
57% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
8
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
14
Sample volume (mL)
1
Orientation
Bottom-up
Speed (g)
100400
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4mL
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
1.857
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
EM
EM-type
Transmission­-EM
Image type
Wide-field
Extra information
TEM was performed for the time course experiment as well, but no images in paper
EV230812 4/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 57%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
57% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
WT 24h
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
2.1
Sample volume (mL)
0.2
Orientation
Bottom-up
Speed (g)
120000
Duration (min)
1020
Fraction volume (mL)
0.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.1
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
TDB
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230812 5/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 57%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
57% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
WT 36h
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
2.1
Sample volume (mL)
0.2
Orientation
Bottom-up
Speed (g)
120000
Duration (min)
1020
Fraction volume (mL)
0.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.1
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
TDB
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230812 6/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 57%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
57% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
WT 48h
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
2.1
Sample volume (mL)
0.2
Orientation
Bottom-up
Speed (g)
120000
Duration (min)
1020
Fraction volume (mL)
0.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.1
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
TDB
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230812 7/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 57%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
57% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
WT 72h
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
235000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
2.1
Sample volume (mL)
0.2
Orientation
Bottom-up
Speed (g)
120000
Duration (min)
1020
Fraction volume (mL)
0.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.1
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
TDB
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230812 8/14 Streptomyces sp. Mg1 PSK0558 (d)(U)C
DG
Filtration
Hoefler BC 2017 57%

Study summary

Full title
All authors
Hoefler BC, Stubbendieck RM, Josyula NK, Moisan SM, Schulze EM, Straight PD
Journal
Cell Chem Biol
Abstract
Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and (show more...)Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology. (hide)
EV-METRIC
57% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
WT 96h
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
not specified
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Streptomyces sp. Mg1
Sample Type
Cell culture supernatant
EV-producing cells
PSK0558
EV-harvesting Medium
Serum free medium
Separation Method