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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210209	6/7	Homo sapiens	VERO-E6	(d)(U)C DG	Xie F	2021	78%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. 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% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA 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	MCF7	(d)(U)C	Xie F	2021	56%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. 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% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA 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	HEK293T	(d)(U)C	Xie F	2021	45%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. 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% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA 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	HEK293T	(d)(U)C	Xie F	2021	45%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. 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% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA 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	HEK293T	(d)(U)C	Xie F	2021	45%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. 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% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA 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	MCF7	(d)(U)C	Xie F	2021	45%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. 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% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA 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	VERO-E6	(d)(U)C	Xie F	2021	45%
Study summary Full title Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. 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% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Detected EV-associated proteins TSG101/ Alix/ ACE2 ELISA 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

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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

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