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

Study summary

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

Study summary

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

Study summary

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

Study summary

Full title
All authors
Vergauwen G, Dhondt B, Van Deun J, De Smedt E, Berx G, Timmerman E, Gevaert K, Miinalainen I, Cocquyt V, Braems G, Van den Broecke R, Denys H, De Wever O, Hendrix A.
Journal
Sci Rep
Abstract
Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust is (show more...)Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Ultrafiltration
Protein markers
EV: Alix/ CD81/ TSG101
non-EV: Argonaute-2/ PMP70
Proteomics
no
EV density (g/ml)
1.094
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MCF7 Rab27b-GFP
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm,
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,…)
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ CD81/ TSG101
Not detected contaminants
Argonaute-2/ PMP70
Characterization: Particle analysis
EV170046 1/2 Sus scrofa Cell culture supernatant DG
dUC
Ultrafiltration
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% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Ultrafiltration
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
Sample Condition
Basolateral side of polarized layer
EV-producing cells
Primary retinal pigmented epithelium cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
Density medium
Iodixanol
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
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
TSG101, Syntenin-1
Not detected contaminants
Calreticulin
Proteomics database
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 Cell culture supernatant DG
dUC
Ultrafiltration
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% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Ultrafiltration
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
Sample Condition
Apical side of polarized layer
EV-producing cells
Primary retinal pigmented epithelium cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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
Density medium
Iodixanol
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
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
TSG101, Syntenin-1
Not detected contaminants
Calreticulin
Proteomics database
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 Cell culture supernatant DG
Filtration
dUC
Ying, Wei 2017 88%

Study summary

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

Study summary

Full title
All authors
Jeurissen S, Vergauwen G, Van Deun J, Lapeire L, Depoorter V, Miinalainen I, Sormunen R, Van den Broecke R, Braems G, Cocquyt V, Denys H, Hendrix A
Journal
Cell Adh Migr
Abstract
Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to (show more...)Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076-1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active. (hide)
EV-METRIC
88% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Adipose tissue
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Adj. k-factor
138.6 (pelleting) / 138.6 (washing)
Protein markers
EV: CD9/ Flotillin-1/ HSP70/ FABP4/ CD63
non-EV: Calreticulin/ GM130/ Prohibitin
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Adipose tissue
Sample Condition
breast cancer
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
138.6
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
138.6
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
1
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
297.9
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1, HSP70, FABP4
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
116
EV concentration
Yes
Particle yield
2.2 10E09
EM
EM-type
Immune-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
EV170028 1/1 Homo sapiens Cell culture supernatant Commercial method Foers AD 2017 77%

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
77% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method
Adj. k-factor
88.25 (pelleting) / 88.25 (washing)
Protein markers
EV: Alix/ CD63/ CD81/ TSG101
non-EV: GM130/ cytochromec
Proteomics
no
Show all info
Study aim
Function, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Colorectal cancer
EV-producing cells
Primary tumor-derived fibroblasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
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 Concentration
100
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, CD81, TSG101
Not detected contaminants
GM130, cytochrome c
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
113
EV concentration
Yes
Particle yield
1.57E+12 particles/million cells
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170063 1/2 Homo sapiens Cell culture supernatant DG
dUC
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% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + 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
Sample Condition
Cyprofloxacin
EV-producing cells
Jurkat
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: 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
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
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 Cell culture supernatant DG
dUC
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% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + 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
Sample Condition
Control condition
EV-producing cells
Jurkat
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: 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
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
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 dUC Maroto, Rosario 2017 75%

Study summary

Full title
All authors
Maroto R, Zhao Y, Jamaluddin M, Popov VL, Wang H, Kalubowilage M, Zhang Y, Luisi J, Sun H, Culbertson CT, Bossmann SH, Motamedi M, Brasier AR
Journal
J Extracell Vesicles
Abstract
Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and (show more...)Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation. (hide)
EV-METRIC
75% (83rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bronchoalveolar lavage fluid
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
60.38 (pelleting) / 60.38 (washing)
Protein markers
EV: CD63/ HSP90/ Alix
non-EV: Grp94/ beta-actin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Bronchoalveolar lavage fluid
Sample Condition
poly IC stimulated
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
60.38
Wash: time (min)
60
Wash: Rotor Type
TLA-100.3
Wash: speed (g)
100000
Wash: adjusted k-factor
60.38
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63,HSP90,Alix
Not detected contaminants
Grp94,beta-actin
Proteomics database
No
Characterization: Particle analysis
DLS
Report type
Median
Reported size (nm)
95
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170061 1/3 Homo sapiens Cell culture supernatant dUC
Filtration
SEC
Ultrafiltration
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% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + SEC + Ultrafiltration
Protein markers
EV: CD63/ CD81/ MFGE8/ CD9
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
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, MFGE8
Flow cytometry specific beads
Selected surface protein(s)
CD9, CD63, CD81
Proteomics database
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 dUC
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% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + SEC
Protein markers
EV: TSG101/ CD81/ 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
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
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 Concentration
6.8
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
TSG101,CD81,CD9
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)
EV170018 1/12 Homo sapiens Cell culture supernatant Commercial method
dUC
Filtration
Gebraad A 2017 57%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + dUC + Filtration
Adj. k-factor
211.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 1, grown on hydroxyapatite
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
126
EV concentration
Yes
Particle yield
2.35E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Extra information
Pelleting by ultracentrifugation was only added to the protocol for EM analysis.
EV170018 3/12 Homo sapiens Cell culture supernatant Commercial method
dUC
Filtration
Gebraad A 2017 57%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + dUC + Filtration
Adj. k-factor
211.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 2, grown on hydroxyapatite
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
121
EV concentration
Yes
Particle yield
6.03E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Extra information
Pelleting by ultracentrifugation was only added to the protocol for EM analysis.
EV170018 5/12 Homo sapiens Cell culture supernatant Commercial method
dUC
Filtration
Gebraad A 2017 57%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + dUC + Filtration
Adj. k-factor
211.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 2, grown on polystyrene
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
126
EV concentration
Yes
Particle yield
1.96E+10 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Extra information
Pelleting by ultracentrifugation was only added to the protocol for EM analysis.
EV170018 7/12 Homo sapiens Cell culture supernatant Commercial method
dUC
Filtration
Gebraad A 2017 57%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + dUC + Filtration
Adj. k-factor
211.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 2, LPS-activated
EV-producing cells
primary circulating monocytes
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
91
EV concentration
Yes
Particle yield
1.68E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Extra information
Pelleting by ultracentrifugation was only added to the protocol for EM analysis.
EV170018 8/12 Homo sapiens Cell culture supernatant Commercial method
dUC
Filtration
Gebraad A 2017 57%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + dUC + Filtration
Adj. k-factor
211.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 1, grown on polystyrene
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
114
EV concentration
Yes
Particle yield
1.17E+10 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Extra information
Pelleting by ultracentrifugation was only added to the protocol for EM analysis.
EV170018 12/12 Homo sapiens Cell culture supernatant Commercial method
dUC
Filtration
Gebraad A 2017 57%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + dUC + Filtration
Adj. k-factor
211.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 1, LPS-activated
EV-producing cells
primary circulating monocytes
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon)
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
107
EV concentration
Yes
Particle yield
1.43E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Extra information
Pelleting by ultracentrifugation was only added to the protocol for EM analysis.
EV170012 1/3 Homo sapiens Cell culture supernatant dUC Gualerzi, Alice 2017 56%

Study summary

Full title
All authors
Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica
Journal
Scientific Reports
Abstract
Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide)
EV-METRIC
56% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
158.5 (pelleting) / 158.5 (washing)
Protein markers
EV: CD9/ CD63/ Flotillin-1
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
adipose tissue mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 55.2 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
158.5
Wash: time (min)
70
Wash: Rotor Type
Type 55.2 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
158.5
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
46.5 ± 15.8
Other particle analysis name(1)
Raman spectroscopy
Extra information
Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA
EV170012 2/3 Homo sapiens Cell culture supernatant dUC Gualerzi, Alice 2017 56%

Study summary

Full title
All authors
Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica
Journal
Scientific Reports
Abstract
Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide)
EV-METRIC
56% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
158.5 (pelleting) / 158.5 (washing)
Protein markers
EV: CD9/ CD63/ Flotillin-1
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
dermal fibroblasts
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 55.2 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
158.5
Wash: time (min)
70
Wash: Rotor Type
Type 55.2 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
158.5
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
46.5 ± 15.8
Other particle analysis name(1)
Raman spectroscopy
Extra information
Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA
EV170012 3/3 Homo sapiens Cell culture supernatant dUC Gualerzi, Alice 2017 56%

Study summary

Full title
All authors
Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica
Journal
Scientific Reports
Abstract
Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide)
EV-METRIC
56% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
158.5 (pelleting) / 158.5 (washing)
Protein markers
EV: CD9/ CD63/ Flotillin-1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
bone marrow-derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 55.2 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
158.5
Wash: time (min)
70
Wash: Rotor Type
Type 55.2 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
158.5
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
46.5 ± 15.8
Other particle analysis name(1)
Raman spectroscopy
Extra information
Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA
EV170060 1/4 Bos taurus Other dUC Kornilov R 2017 55%

Study summary

Full title
All authors
Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S
Journal
J Extracell Vesicles
Abstract
Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide)
EV-METRIC
55% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Other
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: CD71
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Other
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD71
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
1.00E+10 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Extra information
Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).
EV170060 2/4 Bos taurus Serum dUC Kornilov R 2017 55%

Study summary

Full title
All authors
Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S
Journal
J Extracell Vesicles
Abstract
Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide)
EV-METRIC
55% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: CD63/ HSP70/ CD71
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Serum
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, CD71
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
2.50E+11 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Extra information
Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).
EV170060 3/4 Bos taurus Other dUC Kornilov R 2017 55%

Study summary

Full title
All authors
Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S
Journal
J Extracell Vesicles
Abstract
Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide)
EV-METRIC
55% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Other
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: CD63/ HSP70/ CD71
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Other
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, CD71
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
3.00E+10 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Extra information
Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).
EV170060 4/4 Bos taurus Other dUC Kornilov R 2017 55%

Study summary

Full title
All authors
Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S
Journal
J Extracell Vesicles
Abstract
Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide)
EV-METRIC
55% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Other
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: CD63/ HSP70/ CD71
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Other
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, CD71
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
1.00E+10 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
Extra information
Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).
EV170045 1/1 Homo sapiens Cell culture supernatant dUC
Filtration
Treps L 2017 55%

Study summary

Full title
All authors
Treps L, Perret R, Edmond S, Ricard D, Gavard J
Journal
J Extracell Vesicles
Abstract
Glioblastoma multiforme (GBM) are mortifying brain tumours that contain a subpopulation of tumour ce (show more...)Glioblastoma multiforme (GBM) are mortifying brain tumours that contain a subpopulation of tumour cells with stem-like properties, termed glioblastoma stem-like cells (GSCs). GSCs largely contribute to tumour initiation, propagation and resistance to current anti-cancer therapies. GSCs are situated in perivascular niches, closely associated with brain microvascular endothelial cells, thereby involved in bidirectional molecular and cellular interactions. Moreover, extracellular vesicles are suspected to carry essential information that can adapt the microenvironment to the tumour's needs, including tumour-induced angiogenesis. In GBM, extracellular vesicles produced by differentiated tumour cells and GSCs were demonstrated to disseminate locally and at distance. Here, we report that the pro-angiogenic pro-permeability factor VEGF-A is carried in extracellular vesicles secreted from ex vivo cultured patient-derived GSCs. Of note, extracellular vesicle-derived VEGF-A contributes to the in vitro elevation of permeability and angiogenic potential in human brain endothelial cells. Indeed, VEGF-A silencing in GSCs compromised in vitro extracellular vesicle-mediated increase in permeability and angiogenesis. From a clinical standpoint, extracellular vesicles isolated from circulating blood of GBM patients present higher levels of VEGF-A, as compared to healthy donors. Overall, our results suggest that extracellular vesicle-harboured VEGF-A targets brain endothelial cells and might impact their ability to form new vessels. Thus, tumour-released EV cargo might emerge as an instrumental part of the tumour-induced angiogenesis and vascular permeability modus operandi in GBM. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
138.6 (pelleting) / 138.6 (washing)
Protein markers
EV: VEGF-A/ ANXA5/ CD63/ MMP1/ TIMP1/ TIMP2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Brain cancer
EV-producing cells
Primary glioblastoma cells
EV-harvesting Medium
Serum free medium
Cell viability
85
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
240
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
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
ELISA
Lysis buffer provided?
Yes
Flow cytometry
Type of Flow cytometry
MACSQuant Analyzer
Other 1
Protein array
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
109
EV concentration
Yes
EM
EM-type
Transmission-EM/ Immune-EM
Image type
Close-up, Wide-field
Extra information
Wash volume not in original publication.
EV170018 2/12 Homo sapiens Cell culture supernatant dUC
Filtration
Gebraad A 2017 55%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
211.6 (pelleting) / 211.6 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 3, grown on hydroxyapatite
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
120000
Wash: adjusted k-factor
211.6
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
120
EV concentration
Yes
Particle yield
1.20E+08 particles/ml start sample
EV170018 4/12 Homo sapiens Cell culture supernatant dUC
Filtration
Gebraad A 2017 55%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
211.6 (pelleting) / 211.6 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 4, grown on hydroxyapatite
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
120000
Wash: adjusted k-factor
211.6
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
135
EV concentration
Yes
Particle yield
7.90E+08 particles/ml start sample
EV170018 6/12 Homo sapiens Cell culture supernatant dUC
Filtration
Gebraad A 2017 55%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
211.6 (pelleting) / 211.6 (washing)
Protein markers
EV: HSP70/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 3, LPS-activated
EV-producing cells
primary circulating monocytes
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
120000
Wash: adjusted k-factor
211.6
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
133
EV concentration
Yes
Particle yield
3.60E+08 particles/ml start sample
EV170018 9/12 Homo sapiens Cell culture supernatant dUC
Filtration
Gebraad A 2017 55%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
211.6 (pelleting) / 211.6 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 3, grown on polystyrene
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
120000
Wash: adjusted k-factor
211.6
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
134
EV concentration
Yes
Particle yield
7.40E+08 particles/ml start sample
EV170018 10/12 Homo sapiens Cell culture supernatant dUC
Filtration
Gebraad A 2017 55%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
211.6 (pelleting) / 211.6 (washing)
Protein markers
EV: CD63/ HSP70/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 4, grown on polystyrene
EV-producing cells
osteoclasts
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
120000
Wash: adjusted k-factor
211.6
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
131
EV concentration
Yes
Particle yield
5.40E+08 particles/ml start sample
EV170018 11/12 Homo sapiens Cell culture supernatant dUC
Filtration
Gebraad A 2017 55%

Study summary

Full title
All authors
Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R
Journal
FEBS J
Abstract
Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
211.6 (pelleting) / 211.6 (washing)
Protein markers
EV: HSP70/ TSG101/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Donor 4, LPS-activated
EV-producing cells
primary circulating monocytes
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
211.6
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
120000
Wash: adjusted k-factor
211.6
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
147
EV concentration
Yes
Particle yield
3.20E+08 particles/ml start sample
EV170016 2/2 Mus musculus Cell culture supernatant dUC
Filtration
Ying, Wei 2017 55%

Study summary

Full title
All authors
Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, Ofrecio JM, Wollam J, Hernandez-Carretero A, Fu W, Li P, Olefsky JM
Journal
Cell
Abstract
MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we (show more...)MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis. (hide)
EV-METRIC
55% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
256 (pelleting) / 256 (washing)
Protein markers
EV: CD9/ CD63/ HSP70/ TSG101/ Syntenin1
non-EV: Grp94
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
adipose tissue-derived macrophages
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Commercial EDS
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
240-360
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
256.0
Wash: time (min)
20
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
256.0
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
1.13-1.15 g/ml
Used subtypes
Yes
Characterization: Protein analysis
Protein Concentration Method
DC protein assay
Protein Concentration
9-May
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, HSP70, TSG101, Syntenin1
Not detected contaminants
Grp94
Extra information
Full UC protocol not in original article
EV170062 1/1 Homo sapiens Blood plasma Commercial method
dUC
Immunoaffinity capture (non-commercial)
Mustapic M 2017 50%

Study summary

Full title
All authors
Mustapic M, Eitan E, Werner JK, Berkowitz ST, Lazaropoulos MP, Tran J, Goetzl EJ, Kapogiannis D
Journal
J Cell Sci
Abstract
Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin (show more...)Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin from peripheral blood and using them as a biomarker discovery platform for neurological disorders. This methodology has demonstrated excellent diagnostic and predictive performance for Alzheimer's and other neurodegenerative diseases in multiple studies, providing a strong proof of concept for this approach. Here, we describe our methodology in detail and offer further evidence that isolated EVs are enriched for neuronal origin. In addition, we present evidence that EVs enriched for neuronal origin represent a more sensitive and accurate base for biomarkers than plasma, serum, or non-enriched total plasma EVs. Finally, we proceed to investigate the protein content of EVs enriched for neuronal origin and compare it with other relevant enriched and non-enriched populations of plasma EVs. Neuronal-origin enriched plasma EVs contain higher levels of signaling molecules of great interest for cellular metabolism, survival, and repair, which may be useful as biomarkers and to follow response to therapeutic interventions in a mechanism-specific manner. (hide)
EV-METRIC
50% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial method + dUC + Immunoaffinity capture (non-commercial)
Protein markers
EV: CD9/ L1CAM/ MAP2/ gamma-enolase/ pTau23/ tubulin-betaIII/ TSG101/ L1CAMandCD81
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, L1CAM, MAP2, gamma-enolase, pTau23, tubulin-betaIII
ELISA
Lysis buffer provided?
Yes
Other 1
Proteome Profiler Human Phospho-Mitogen-activated Protein Kinase (MAPK) Antibody Array (Catalog # ARY002B), Human Kidney Biomarker Antibody Array (Catalog # ARY019), Human Phospho-Kinase Antibody Arra
Characterization: Particle analysis
PMID previous EV particle analysis
Nanoparticle tracking analysis
Extra particle analysis
NTA
Report type
Size range/distribution
EV concentration
Yes
EM
EM-type
Immune-EM
Image type
Close-up, Wide-field
EV170061 2/3 Homo sapiens Cell culture supernatant dUC Benedikter BJ 2017 50%

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
50% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
133.2 (pelleting) / 48.17 (washing)
Protein markers
EV: CD63/ CD81
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
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2
Wash: time (min)
150
Wash: Rotor Type
NVT 90
Wash: speed (g)
110656
Wash: adjusted k-factor
48.17
Characterization: Protein analysis
Protein Concentration Method
Bradford
Flow cytometry specific beads
Selected surface protein(s)
CD63
Proteomics database
No
Characterization: Particle analysis
TRPS
EV concentration
Yes
Particle yield
2.00E+00
EV170061 3/3 Homo sapiens Cell culture supernatant dUC Benedikter BJ 2017 50%

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
50% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
133.2 (pelleting)
Protein markers
EV: CD63/ CD81
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
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2