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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV220127 1/2 Homo sapiens lung tissue (d)(U)C
DG
Filtration
Liu, Bowen/ Jin, Yuan 2022 100%

Study summary

Full title
All authors
Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide)
EV-METRIC
100% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
lung tissue
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
20553 (pelleting) / 17842 (washing)
Protein markers
EV: Alix/ CD9/ CD81
non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.1
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
lung tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
20553
Wash: volume per pellet (ml)
1.5
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
110,000
Wash: adjusted k-factor
17842
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
0.25 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.45
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
180,000
Duration (min)
780
Fraction volume (mL)
0.45
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
17842
Pelleting-wash: volume per pellet (mL)
1.5
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
TLA-55
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
0.8
Western Blot
Detected EV-associated proteins
Alix/ CD9/ CD81
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
131.7
Particle analysis: flow cytometry
Flow cytometer type
BD LSRFortessa
Hardware adjustment
use calibration beads
Calibration bead size
0.05/ 0.1/ 0.2/ 0.3/ 0.5
Report type
Size range/distribution
Reported size (nm)
100 - 200
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV220127 2/2 Mus musculus lung tissue (d)(U)C
DG
Filtration
Liu, Bowen/ Jin, Yuan 2022 100%

Study summary

Full title
All authors
Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide)
EV-METRIC
100% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
lung tissue
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
20553 (pelleting) / 17842 (washing)
Protein markers
EV: Alix/ CD9/ Flotillin-1/ TSG101
non-EV: GM130/ Calnexin/ Albumin/ Argonaute-2/ Calreticulin/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.1
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
lung tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
20553
Wash: volume per pellet (ml)
1.5
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
110,000
Wash: adjusted k-factor
17842
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
0.25 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.45
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
180,000
Duration (min)
780
Fraction volume (mL)
0.45
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
17842
Pelleting-wash: volume per pellet (mL)
1.5
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
TLA-55
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
0.7
Western Blot
Detected EV-associated proteins
Alix/ CD9/ Flotillin-1/ TSG101
Not detected contaminants
GM130/ Calnexin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
124.9
Particle analysis: flow cytometry
Flow cytometer type
BD LSRFortessa
Hardware adjustment
use calibration beads
Calibration bead size
0.05/ 0.1/ 0.2/ 0.3/ 0.5
Report type
Size range/distribution
Reported size (nm)
100 - 200
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210154 1/2 Homo sapiens human invasive proliferative extravillous cytotrophoblast (HIPEC) DG
(d)(U)C
Bergamelli M 2022 100%

Study summary

Full title
All authors
Bergamelli M, Martin H, Aubert Y, Mansuy JM, Marcellin M, Burlet-Schiltz O, Hurbain I, Raposo G, Izopet J, Fournier T, Benchoua A, Bénard M, Groussolles M, Cartron G, Tanguy Le Gac Y, Moinard N, D'Angelo G, Malnou CE
Journal
Viruses
Abstract
Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pre (show more...)Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other/ small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Alix/ CD63/ CD9/ CD81
non-EV: calnexin/ TOM20
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human invasive proliferative extravillous cytotrophoblast (HIPEC)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell count
1,00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1,7
Fraction processing
Centrifugation
Pelleting: volume per fraction
30
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
30
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD63
Not detected contaminants
calnexin/ TOM20
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
Particle analysis: flow cytometry
Flow cytometer type
Mascquant VYB Myltenyi
Hardware adjustment
Mascquant VYB Myltenyi set up fluorescent beads SSC Megamix, with calibration on gates of 160nm 200nm 250nm and 500nm
Calibration bead size
0.16/ 0.2/ 0.25/ 0.5
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
EM
EM-type
Immuno-EM/ Transmission-EM
EM protein
CD9/ CD81/ CD63
Image type
Close-up, Wide-field
Report size (nm)
120
EV210154 2/2 Homo sapiens human invasive proliferative extravillous cytotrophoblast (HIPEC) DG
(d)(U)C
Bergamelli M 2022 100%

Study summary

Full title
All authors
Bergamelli M, Martin H, Aubert Y, Mansuy JM, Marcellin M, Burlet-Schiltz O, Hurbain I, Raposo G, Izopet J, Fournier T, Benchoua A, Bénard M, Groussolles M, Cartron G, Tanguy Le Gac Y, Moinard N, D'Angelo G, Malnou CE
Journal
Viruses
Abstract
Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pre (show more...)Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
infected by Human Cytomegalovirus / clinical strain VHL/E
Focus vesicles
Other/ small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Alix/ CD63/ CD9/ CD81
non-EV: calnexin/ TOM20
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human invasive proliferative extravillous cytotrophoblast (HIPEC)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell count
1,00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1,7
Fraction processing
Centrifugation
Pelleting: volume per fraction
30
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
30
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix/ CD81
Not detected contaminants
calnexin/ TOM20
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
Particle analysis: flow cytometry
Flow cytometer type
Macsquant VYB Myltenyi
Hardware adjustment
Mascquant VYB Myltenyi set up fluorescent beads SSC Megamix, with calibration on gates of 160nm 200nm 250nm and 500nm
Calibration bead size
0.16/ 0.2/ 0.25/ 0.5
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
EM
EM-type
Immuno-EM/ Transmission-EM
EM protein
CD9/ CD81/ CD63
Image type
Close-up, Wide-field
Report size (nm)
110
EV210151 1/8 Homo sapiens hiPSC (IMR90)-4 DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Flotillin2
Not detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 2.23E7 +- 1.35E7
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 2/8 Homo sapiens CPC (IMR90)-4 DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CPC (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Flotillin2/ TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 8.27E6 +- 3.53E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 3/8 Homo sapiens CMi (IMR90)-4 DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMi (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Flotillin2/ TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 2.95E7 +- 1.19E7
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 4/8 Homo sapiens CMm (IMR90)-4 DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMm (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Flotillin2
Not detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250 nm
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 4.10E7 +- 9.75E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200030 1/2 Homo sapiens oral mucosa lamina propria progenitor cells (d)(U)C
Other/ ExoSpin
UF
Filtration
DG
Knight R 2022 100%

Study summary

Full title
All authors
Knight R, Board-Davies E, Brown H, Clayton A, Davis T, Karatas B, Burston J, Tabi Z, Falcon-Perez JM, Paisey S, Stephens P
Journal
Stem Cells Transl Med
Abstract
Scar formation during wound repair can be devastating for affected individuals. Our group previously (show more...)Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings/ hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other/ small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Commercial method
Ultrafiltration
Filtration
Density gradient
Protein markers
EV: CD81/ CD63/ CD9
non-EV: CD105/ CD90/ CD166
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
oral mucosa lamina propria progenitor cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0.2M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
16
Fraction volume (mL)
0.3
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Commercial kit
Other/ ExoSpin
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry aspecific beads
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected contaminants
CD90/ CD105/ CD166
Flow cytometry specific beads
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
94
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 7.7E+12
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV200030 2/2 Homo sapiens bone marrow derived mesenchymal stromal cells (d)(U)C
Other/ ExoSpin
UF
Filtration
DG
Knight R 2022 100%

Study summary

Full title
All authors
Knight R, Board-Davies E, Brown H, Clayton A, Davis T, Karatas B, Burston J, Tabi Z, Falcon-Perez JM, Paisey S, Stephens P
Journal
Stem Cells Transl Med
Abstract
Scar formation during wound repair can be devastating for affected individuals. Our group previously (show more...)Scar formation during wound repair can be devastating for affected individuals. Our group previously documented the therapeutic potential of novel progenitor cell populations from the non-scarring buccal mucosa. These Oral Mucosa Lamina Propria-Progenitor Cells (OMLP-PCs) are multipotent, immunosuppressive, and antibacterial. Small extracellular vesicles (sEVs) may play important roles in stem cell-mediated repair in varied settings/ hence, we investigated sEVs from this source for wound repair. We created an hTERT immortalized OMLP-PC line (OMLP-PCL) and confirmed retention of morphology, lineage plasticity, surface markers, and functional properties. sEVs isolated from OMLP-PCL were analyzed by nanoparticle tracking analysis, Cryo-EM and flow cytometry. Compared to bone marrow-derived mesenchymal stromal cells (BM-MSC) sEVs, OMLP-PCL sEVs were more potent at driving wound healing functions, including cell proliferation and wound repopulation and downregulated myofibroblast formation. A reduced scarring potential was further demonstrated in a preclinical in vivo model. Manipulation of OMLP-PCL sEVs may provide novel options for non-scarring wound healing in clinical settings. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other/ small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Commercial method
Ultrafiltration
Filtration
Density gradient
Protein markers
EV: CD81/ CD63/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
bone marrow derived mesenchymal stromal cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0.2M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
16
Fraction volume (mL)
0.3
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Commercial kit
Other/ ExoSpin
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry aspecific beads
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected contaminants
CD90/ CD105/ CD166
Flow cytometry specific beads
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
99.5
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 4.27E+12
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV200110  1/3 Homo sapiens NCI-H1975 (d)(U)C
DG
Van Hoof R 2022 89%

Study summary

Full title
All authors
Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV: Calnexin/ Ribosomal protein S6
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
NCI-H1975
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.7
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
366613
Duration (min)
960
Fraction volume (mL)
0.48
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
HSP70/ CD81
Detected contaminants
Ribosomal protein S6
Not detected contaminants
Calnexin
Flow cytometry
Type of Flow cytometry
BD Influx flow cytometer
Hardware adaptation to ~100nm EV's
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
0.1/ 0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
Other/ RNase A/T1 mix
RNAse concentration
RNase A: 0.02 mg/ml - RNase T1: 50 U/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
202
EV concentration
Yes
Particle yield
as number of particles per mililiter in the final EV fraction (500 l)/ other:: 5.00E+10
Particle analysis: flow cytometry
Flow cytometer type
BD Influx flow cytometer
Hardware adjustment
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
1
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 5/8 Homo sapiens hiPSC (DF19-9-11T.H) DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
90
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 1.99E7 +- 1.99E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 6/8 Homo sapiens CPC (DF19-9-11T.H) DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CPC (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 6.43E06 +- 5.40E05
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 7/8 Homo sapiens CMi (DF19-9-11T.H) DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMi (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 3.49E07 +- 2.85E06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 8/8 Homo sapiens CMm (DF19-9-11T.H) DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMm (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
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)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 2.13E07 +- 2.25E06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV21008 1/2 Homo sapiens Blood plasma DG
(d)(U)C
Annalisa Radeghieri 2022 89%

Study summary

Full title
All authors
Annalisa Radeghieri, Silvia Alacqua, Andrea Zendrini, Vanessa Previcini, Francesca Todaro, Giuliana Martini, Doris Ricotta, Paolo Bergese
Journal
Journal of Extracellular Biology
Abstract
Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clott (show more...)Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the “EV-protein corona.” The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Adam 10/ Alix/ Antithrombin 3
non-EV: Argonaute2/ Apo A1/ GM130
Proteomics
no
EV density (g/ml)
1.11-1.22
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
8%
Highest density fraction
75%
Total gradient volume, incl. sample (mL)
4,8
Sample volume (mL)
1
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0,4
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Adam 10/ TSG101/ Alix/ CD81
Not detected contaminants
Apo A1/ GM130
Detected EV-associated proteins
CD63/ TSG101
Detected contaminants
Argonaute2
Detected EV-associated proteins
Antithrombin 3
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Atomic force-EM
Image type
Close-up, Wide-field
Report size (nm)
50
EV210024 3/12 Homo sapiens Glioblastoma Stem-like cells (GSC) (d)(U)C
DG
André-Grégoire, Gwennan 2022 89%

Study summary

Full title
All authors
Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard
Journal
iScience
Abstract
Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: Alix/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Glioblastoma Stem-like cells (GSC)
EV-harvesting Medium
Serum free medium
Cell count
5.00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
11
Wash: time (min)
120
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40
Total gradient volume, incl. sample (mL)
11.5
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
120
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ Alix
Not detected contaminants
GM130
ELISA
Detected EV-associated proteins
CD63
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50
EV200110  2/3 Homo sapiens NCI-H1975 (d)(U)C
UF
qEV
DG
Van Hoof R 2022 88%

Study summary

Full title
All authors
Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Ultrafiltration
Commercial method
Density gradient
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV: Calnexin/ Ribosomal protein S6
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
NCI-H1975
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.7
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
366613
Duration (min)
960
Fraction volume (mL)
0.48
Fraction processing
None
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
HSP70/ CD81
Not detected contaminants
Calnexin/ Ribosomal protein S6
Flow cytometry
Type of Flow cytometry
BD Influx flow cytometer
Hardware adaptation to ~100nm EV's
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
0.1/ 0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
Other/ RNase A/T1 mix
RNAse concentration
RNase A: 0.02 mg/ml - RNase T1: 50 U/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
186
EV concentration
Yes
Particle yield
as number of particles per mililiter in the final EV fraction (500 l)/ other:: 1.70E+10
Particle analysis: flow cytometry
Flow cytometer type
BD Influx flow cytometer
Hardware adjustment
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
1
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200110  3/3 Homo sapiens NCI-H1975 (d)(U)C
Other/ exoEasy
Filtration
DG
Van Hoof R 2022 88%

Study summary

Full title
All authors
Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Commercial method
Filtration
Density gradient
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV: Calnexin/ Ribosomal protein S6
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
NCI-H1975
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.7
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
366613
Duration (min)
960
Fraction volume (mL)
0.48
Fraction processing
None
Filtration steps
> 0.45 µm, 0.22µm or 0.2µm
Commercial kit
Other/ exoEasy
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
HSP70/ CD81
Not detected contaminants
Calnexin/ Ribosomal protein S6
Flow cytometry
Type of Flow cytometry
BD Influx flow cytometer
Hardware adaptation to ~100nm EV's
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
0.1/ 0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
Other/ RNase A/T1 mix
RNAse concentration
RNase A: 0.02 mg/ml - RNase T1: 50 U/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
250
EV concentration
Yes
Particle yield
as number of particles per mililiter in the final EV fraction (500 l)/ other:: 2.60E+11
Particle analysis: flow cytometry
Flow cytometer type
BD Influx flow cytometer
Hardware adjustment
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
1
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210213 1/6 Homo sapiens MDA-MB-231-luc-D3H1 DG
(d)(U)C
Lischnig A 2022 88%

Study summary

Full title
All authors
Lischnig A, Bergqvist M, Ochiya T, Lässer C
Journal
Mol Cell Proteomics
Abstract
There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs (show more...)There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Large extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231-luc-D3H1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
37.50%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Bottom-up
Speed (g)
180000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1
Not detected EV-associated proteins
CD81/ CD63
Detected contaminants
Calnexin
Proteomics database
Yes:
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 1.45E+07
EM
EM-type
Transmission-EM
Image type
Close-up
EV210213 2/6 Homo sapiens MDA-MB-231-luc-D3H1 DG
(d)(U)C
Lischnig A 2022 88%

Study summary

Full title
All authors
Lischnig A, Bergqvist M, Ochiya T, Lässer C
Journal
Mol Cell Proteomics
Abstract
There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs (show more...)There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231-luc-D3H1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
118000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
37.50%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Bottom-up
Speed (g)
180000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1
Not detected EV-associated proteins
CD81/ CD63
Detected contaminants
Calnexin
Proteomics database
Yes:
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 1.45E+07
EM
EM-type
Transmission-EM
Image type
Close-up
EV210213 3/6 Homo sapiens MDA-MB-231-luc-D3H2LN DG
(d)(U)C
Lischnig A 2022 88%

Study summary

Full title
All authors
Lischnig A, Bergqvist M, Ochiya T, Lässer C
Journal
Mol Cell Proteomics
Abstract
There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs (show more...)There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Large extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231-luc-D3H2LN
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
37.50%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Bottom-up
Speed (g)
180000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63
Not detected EV-associated proteins
CD81
Not detected contaminants
Calnexin
Proteomics database
Yes:
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 8.90E+06
EM
EM-type
Transmission-EM
Image type
Close-up
EV210213 4/6 Homo sapiens MDA-MB-231-luc-D3H2LN DG
(d)(U)C
Lischnig A 2022 88%

Study summary

Full title
All authors
Lischnig A, Bergqvist M, Ochiya T, Lässer C
Journal
Mol Cell Proteomics
Abstract
There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs (show more...)There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231-luc-D3H2LN
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
118000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
37.50%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Bottom-up
Speed (g)
180000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63
Not detected EV-associated proteins
CD81
Not detected contaminants
Calnexin
Proteomics database
Yes:
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 8.90E+06
EM
EM-type
Transmission-EM
Image type
Close-up
EV210213 5/6 Homo sapiens MDA-MB-231-luc-BMD2a DG
(d)(U)C
Lischnig A 2022 88%

Study summary

Full title
All authors
Lischnig A, Bergqvist M, Ochiya T, Lässer C
Journal
Mol Cell Proteomics
Abstract
There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs (show more...)There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other/ Large extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231-luc-BMD2a
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
37.50%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Bottom-up
Speed (g)
180000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Detected contaminants
Calnexin
Proteomics database
Yes:
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 2.30E+07
EM
EM-type
Transmission-EM
Image type
Close-up
EV210213 6/6 Homo sapiens MDA-MB-231-luc-BMD2a DG
(d)(U)C
Lischnig A 2022 88%

Study summary

Full title
All authors
Lischnig A, Bergqvist M, Ochiya T, Lässer C
Journal
Mol Cell Proteomics
Abstract
There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs (show more...)There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag-liquid chromatography-tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231-luc-BMD2a
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
118000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
37.50%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Bottom-up
Speed (g)
180000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Detected contaminants
Calnexin
Proteomics database
Yes:
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 2.30E+07
EM
EM-type
Transmission-EM
Image type
Close-up
EV180067 1/5 Mus musculus MPI cells UF
(d)(U)C
qEV
DG
Deville S 2022 88%

Study summary

Full title
All authors
Deville S, Garcia Romeu H, Oeyen E, Mertens I, Nelissen I, Salvati A
Journal
Int J Mol Sci
Abstract
Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in (show more...)Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
(Differential) (ultra)centrifugation
Commercial method
Density gradient
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9
non-EV: Cytochrome C/ GRP94
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
MPI cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.8
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1
Not detected contaminants
Cytochrome C/ GRP94
Flow cytometry
Type of Flow cytometry
BD Influx instrument/ CytoFlex
Hardware adaptation to ~100nm EV's
Both BD Influx instrument (with a small particle detector) and Beckman Coulter CytoFlex instrument have been validated for the measurement of EVs.
Calibration bead size
0.1-0.9 µm beads
Detected EV-associated proteins
CD9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
116
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection.
Calibration bead size
0.1
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV180067 2/5 Mus musculus MPI cells UF
(d)(U)C
qEV
DG
Deville S 2022 88%

Study summary

Full title
All authors
Deville S, Garcia Romeu H, Oeyen E, Mertens I, Nelissen I, Salvati A
Journal
Int J Mol Sci
Abstract
Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in (show more...)Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
LPS
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
(Differential) (ultra)centrifugation
Commercial method
Density gradient
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9
non-EV: Cytochrome C/ GRP94
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
MPI cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.8
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1
Not detected contaminants
Cytochrome C/ GRP94
Flow cytometry
Type of Flow cytometry
BD Influx instrument/ CytoFlex
Hardware adaptation to ~100nm EV's
Both BD Influx instrument (with a small particle detector) and Beckman Coulter CytoFlex instrument have been validated for the measurement of EVs.
Calibration bead size
0.1-0.9 µm beads
Detected EV-associated proteins
CD9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
114
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV180067 3/5 Mus musculus MPI cells UF
(d)(U)C
qEV
DG
Deville S 2022 88%

Study summary

Full title
All authors
Deville S, Garcia Romeu H, Oeyen E, Mertens I, Nelissen I, Salvati A
Journal
Int J Mol Sci
Abstract
Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in (show more...)Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NH2-PS NPs
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
(Differential) (ultra)centrifugation
Commercial method
Density gradient
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9
non-EV: Cytochrome C/ GRP94
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
MPI cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.8
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1
Not detected contaminants
Cytochrome C/ GRP94
Flow cytometry
Type of Flow cytometry
BD Influx instrument/ CytoFlex
Hardware adaptation to ~100nm EV's
Both BD Influx instrument (with a small particle detector) and Beckman Coulter CytoFlex instrument have been validated for the measurement of EVs.
Calibration bead size
0.1-0.9 µm beads
Detected EV-associated proteins
CD9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
139
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
Image type
Close-up, Wide-field
EV180067 4/5 Mus musculus MPI cells UF
(d)(U)C
qEV
DG
Deville S 2022 88%

Study summary

Full title
All authors
Deville S, Garcia Romeu H, Oeyen E, Mertens I, Nelissen I, Salvati A
Journal
Int J Mol Sci
Abstract
Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in (show more...)Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
COOH-PS NPs
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
(Differential) (ultra)centrifugation
Commercial method
Density gradient
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9
non-EV: Cytochrome C/ GRP94
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
MPI cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.8
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1
Not detected contaminants
GRP94/ Cytochrome C
Flow cytometry
Type of Flow cytometry
BD Influx instrument/ CytoFlex
Hardware adaptation to ~100nm EV's
Both BD Influx instrument (with a small particle detector) and Beckman Coulter CytoFlex instrument have been validated for the measurement of EVs.
Calibration bead size
0.1-0.9 µm beads
Detected EV-associated proteins
CD9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
112
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV180067 5/5 Mus musculus MPI cells UF
(d)(U)C
qEV
DG
Deville S 2022 88%

Study summary

Full title
All authors
Deville S, Garcia Romeu H, Oeyen E, Mertens I, Nelissen I, Salvati A
Journal
Int J Mol Sci
Abstract
Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in (show more...)Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
SiO2 NPs
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
(Differential) (ultra)centrifugation
Commercial method
Density gradient
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9
non-EV: Cytochrome C/ GRP94
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
MPI cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.8
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1
Not detected contaminants
GRP94/ Cytochrome c
Flow cytometry
Type of Flow cytometry
BD Influx instrument/ CytoFlex
Hardware adaptation to ~100nm EV's
Both BD Influx instrument (with a small particle detector) and Beckman Coulter CytoFlex instrument have been validated for the measurement of EVs.
Calibration bead size
0.1-0.9 µm beads
Detected EV-associated proteins
CD9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
102
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
Calibration bead size
0.1
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210204 1/12 Homo sapiens PANC-1 (d)(U)C
Filtration
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PANC-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ ANXA5/ EpCAM/ ICAM/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
180.8
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 2/12 Homo sapiens PANC-1 (d)(U)C
Filtration
UF
qEV
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Commercial method
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PANC-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ ICAM/ EpCAM/ ANXA5/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 4/12 Homo sapiens PPCL-68 (d)(U)C
Filtration
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Pancreas cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ ANXA5/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ CD63
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PPCL-68
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ ANXA5/ EpCAM/ ICAM/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
180.8
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210204 5/12 Homo sapiens PPCL-68 (d)(U)C
Filtration
UF
qEV
Hinzman CP 2022 78%

Study summary

Full title
All authors
Hinzman CP, Singh B, Bansal S, Li Y, Iliuk A, Girgis M, Herremans KM, Trevino JG, Singh VK, Banerjee PP, Cheema AK
Journal
J Extracell Vesicles
Abstract
Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promotin (show more...)Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomole