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You searched for: EV210209 (EV-TRACK ID)

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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 code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210209 6/7 Homo sapiens Cell culture supernatant (d)(U)C
DG
Xie F 2021 78%

Study summary

Full title
All authors
Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F
Journal
Adv Mater
Abstract
Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: Alix/ TSG101/ ACE2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
VERO-E6
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 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)
120000
Wash: volume per pellet (ml)
36mL/ultracentrifuge tube
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
60%
Orientation
Top-down
Speed (g)
100,000g
Duration (min)
1080
Other
Name other separation method
Density gradient
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
30-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
TSG101/ Alix/ ACE2
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
ACE2
Fluorescent NTA
Antibody details provided?
No
Detected EV-associated proteins
ACE2-GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Used for determining EV concentration?
Yes
EM
EM-type
Transmission-EM/ Immuno-EM
EM protein
ACE2/ TSG101
Image type
Close-up
EV210209 4/7 Homo sapiens Cell culture supernatant (d)(U)C Xie F 2021 56%

Study summary

Full title
All authors
Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F
Journal
Adv Mater
Abstract
Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide)
EV-METRIC
56% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ TSG101/ ACE2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 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)
120000
Wash: volume per pellet (ml)
36mL/ultracentrifuge tube
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
30-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
TSG101/ Alix/ ACE2
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
ACE2
Fluorescent NTA
Antibody details provided?
No
Detected EV-associated proteins
ACE2-GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Used for determining EV concentration?
Yes
EM
EM-type
Immuno-EM
EM protein
ACE2
Image type
Close-up
EV210209 1/7 Homo sapiens Cell culture supernatant (d)(U)C Xie F 2021 45%

Study summary

Full title
All authors
Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F
Journal
Adv Mater
Abstract
Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide)
EV-METRIC
45% (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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ TSG101/ ACE2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HEK293T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 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)
120000
Wash: volume per pellet (ml)
36mL/ultracentrifuge tube
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
30-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
TSG101/ Alix/ ACE2
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
ACE2
Fluorescent NTA
Antibody details provided?
No
Detected EV-associated proteins
ACE2-GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Used for determining EV concentration?
Yes
EV210209 2/7 Homo sapiens Cell culture supernatant (d)(U)C Xie F 2021 45%

Study summary

Full title
All authors
Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F
Journal
Adv Mater
Abstract
Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide)
EV-METRIC
45% (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
Cell culture supernatant
Sample origin
Infected by SeV or VSV
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ TSG101/ ACE2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HEK293T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 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)
120000
Wash: volume per pellet (ml)
36mL/ultracentrifuge tube
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
30-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
TSG101/ Alix/ ACE2
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
ACE2
Fluorescent NTA
Antibody details provided?
No
Detected EV-associated proteins
ACE2-GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Used for determining EV concentration?
Yes
EV210209 3/7 Homo sapiens Cell culture supernatant (d)(U)C Xie F 2021 45%

Study summary

Full title
All authors
Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F
Journal
Adv Mater
Abstract
Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide)
EV-METRIC
45% (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
Cell culture supernatant
Sample origin
ACE2-overexpressing
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ TSG101/ ACE2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HEK293T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 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)
120000
Wash: volume per pellet (ml)
36mL/ultracentrifuge tube
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
30-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
TSG101/ Alix/ ACE2
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
ACE2
Fluorescent NTA
Antibody details provided?
No
Detected EV-associated proteins
ACE2-GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Used for determining EV concentration?
Yes
EV210209 5/7 Homo sapiens Cell culture supernatant (d)(U)C Xie F 2021 45%

Study summary

Full title
All authors
Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F
Journal
Adv Mater
Abstract
Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide)
EV-METRIC
45% (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
Cell culture supernatant
Sample origin
Infected by SeV or VSV
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ TSG101/ ACE2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 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)
120000
Wash: volume per pellet (ml)
36mL/ultracentrifuge tube
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
30-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
TSG101/ Alix/ ACE2
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
ACE2
Fluorescent NTA
Antibody details provided?
No
Detected EV-associated proteins
ACE2-GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Used for determining EV concentration?
Yes
EV210209 7/7 Homo sapiens Cell culture supernatant (d)(U)C Xie F 2021 45%

Study summary

Full title
All authors
Xie F, Su P, Pan T, Zhou X, Li H, Huang H, Wang A, Wang F, Huang J, Yan H, Zeng L, Zhang L, Zhou F
Journal
Adv Mater
Abstract
Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly inte (show more...)Angiotensin converting enzyme 2 (ACE2) is a key receptor present on cell surfaces that directly interacts with the viral spike (S) protein of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It is proposed that inhibiting this interaction can be promising in treating COVID-19. Here, the presence of ACE2 in extracellular vesicles (EVs) is reported and the EV-ACE2 levels are determined by protein palmitoylation. The Cys141 and Cys498 residues on ACE2 are S-palmitoylated by zinc finger DHHC-Type Palmitoyltransferase 3 (ZDHHC3) and de-palmitoylated by acyl protein thioesterase 1 (LYPLA1), which is critical for the membrane-targeting of ACE2 and their EV secretion. Importantly, by fusing the S-palmitoylation-dependent plasma membrane (PM) targeting sequence with ACE2, EVs enriched with ACE2 on their surface (referred to as PM-ACE2-EVs) are engineered. It is shown that PM-ACE2-EVs can bind to the SARS-CoV-2 S-RBD with high affinity and block its interaction with cell surface ACE2 in vitro. PM-ACE2-EVs show neutralization potency against pseudotyped and authentic SARS-CoV-2 in human ACE2 (hACE2) transgenic mice, efficiently block viral load of authentic SARS-CoV-2, and thus protect host against SARS-CoV-2-induced lung inflammation. The study provides an efficient engineering protocol for constructing a promising, novel biomaterial for application in prophylactic and therapeutic treatments against COVID-19. (hide)
EV-METRIC
45% (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
Cell culture supernatant
Sample origin
ACE2-overexpressing stable cell line
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ TSG101/ ACE2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
VERO-E6
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 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)
120000
Wash: volume per pellet (ml)
36mL/ultracentrifuge tube
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
120000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
30-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
TSG101/ Alix/ ACE2
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
ACE2
Fluorescent NTA
Antibody details provided?
No
Detected EV-associated proteins
ACE2-GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Used for determining EV concentration?
Yes
1 - 7 of 7
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210209
species
Homo
sapiens
sample type
Cell
culture
cell type
VERO-E6
MCF7
HEK293T
HEK293T
HEK293T
MCF7
VERO-E6
condition
Control
condition
Control
condition
Control
condition
Infected
by
SeV
or
VSV
ACE2-overexpressing
Infected
by
SeV
or
VSV
ACE2-overexpressing
stable
cell
line
separation protocol
dUC/
Density
gradient
dUC
dUC
dUC
dUC
dUC
dUC
Exp. nr.
6
4
1
2
3
5
7
EV-METRIC %
78
56
45
45
45
45
45