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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV240045	5/6	Homo sapiens	HEK293T	Filtration UF	Pauwels J	2025	75%
Study summary Full title Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics. All authors Pauwels J, Van de Steene T, Van de Velde J, De Muyer F, De Pauw D, Baeke F, Eyckerman S, Gevaert K Journal Mol Cell Proteomics Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the ex (show more...)Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Gag-eGFP OE 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 Filtration Ultrafiltration Protein markers EV: Gag-eGFP non-EV: bovine proteins Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Filtration steps 0.2 or 0.22µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Gag-eGFP Not detected contaminants Calnexin Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 128 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+08 EM EM-type Scanning-EM/ Transmission-EM Image type Close-up, Wide-field

	EV240045	6/6	Homo sapiens	HEK293T	Filtration UF Tween wash	Pauwels J	2025	75%
Study summary Full title Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics. All authors Pauwels J, Van de Steene T, Van de Velde J, De Muyer F, De Pauw D, Baeke F, Eyckerman S, Gevaert K Journal Mol Cell Proteomics Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the ex (show more...)Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Gag-eGFP OE 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 Filtration Ultrafiltration Tween wash Protein markers EV: Gag-eGFP non-EV: bovine proteins Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Filtration steps 0.2 or 0.22µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Other Name other separation method Tween wash Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Gag-eGFP Not detected contaminants Calnexin Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 156 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.30E+07 EM EM-type Scanning-EM/ Transmission-EM Image type Close-up, Wide-field

	EV240164	2/5	Homo sapiens	HeLa	(d)(U)C DG	Xiong J	2025	56%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 56% (89th 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 Japanese encephalitis virus-infected 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: CD63/ CD81 non-EV: Calnexin Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 150-300 EM EM-type Transmission-EM Image type Wide-field

	EV240164	3/5	Sus scrofa domesticus	PIEC	(d)(U)C DG	Xiong J	2025	56%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 56% (89th 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 Japanese encephalitis virus-infected 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: TSG101/ CD81 non-EV: Calnexin Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Cell culture supernatant EV-producing cells PIEC EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 80 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 Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins TSG101/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 150-300 EM EM-type Transmission-EM Image type Wide-field

	EV240164	4/5	Mesocricetus auratus	BHK-21	(d)(U)C DG	Xiong J	2025	56%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 56% (89th 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 Japanese encephalitis virus-infected 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/ Alix non-EV: Calnexin Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Mesocricetus auratus Sample Type Cell culture supernatant EV-producing cells BHK-21 EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 80 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 Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD81/ Alix Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 150-300 EM EM-type Transmission-EM Image type Wide-field

	EV250039	1/2	Homo sapiens	Stool	DG Filtration (d)(U)C SEC (commercial) UF	Mishra S	2025	50%
Study summary Full title Gut microbiome-derived bacterial extracellular vesicles in patients with solid tumours. All authors Mishra S, Tejesvi MV, Hekkala J, Turunen J, Kandikanti N, Kaisanlahti A, Suokas M, Leppä S, Vihinen P, Kuitunen H, Sunela K, Koivunen J, Jukkola A, Kalashnikov I, Auvinen P, Kääriäinen OS, Peñate Medina T, Peñate Medina O, Saarnio J, Meriläinen S, Rautio T, Aro R, Häivälä R, Suojanen J, Laine M, Erawijattari PP, Lahti L, Karihtala P, Ruuska TS, Reunanen J Journal J Adv Res Abstract Gut microbiome-derived nanoparticles, known as bacterial extracellular vesicles (bEVs), have garnere (show more...)Gut microbiome-derived nanoparticles, known as bacterial extracellular vesicles (bEVs), have garnered interest as promising tools for studying the link between the gut microbiome and human health. The diverse composition of bEVs, including their proteins, mRNAs, metabolites, and lipids, makes them useful for investigating diseases such as cancer. However, conventional approaches for studying gut microbiome composition alone may not be accurate in deciphering host-gut microbiome communication. In clinical microbiome research, there is a gap in the knowledge on the role of bEVs in solid tumor patients. (hide) EV-METRIC 50% (76th 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 Stool Sample origin Patients with solid tumor 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 Density gradient Filtration (Differential) (ultra)centrifugation Size-exclusion chromatography (commercial) Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) - Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Stool Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 0.2 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 100000 Filtration steps Larger than 0.45 µm/ Between 0.22 and 0.45 µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Other Name other separation method Size-exclusion chromatography (commercial) Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 202 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.08E+08 EM EM-type Transmission-EM Image type Wide-field

	EV250039	2/2	Homo sapiens	Stool	DG Filtration (d)(U)C SEC (commercial)	Mishra S	2025	50%
Study summary Full title Gut microbiome-derived bacterial extracellular vesicles in patients with solid tumours. All authors Mishra S, Tejesvi MV, Hekkala J, Turunen J, Kandikanti N, Kaisanlahti A, Suokas M, Leppä S, Vihinen P, Kuitunen H, Sunela K, Koivunen J, Jukkola A, Kalashnikov I, Auvinen P, Kääriäinen OS, Peñate Medina T, Peñate Medina O, Saarnio J, Meriläinen S, Rautio T, Aro R, Häivälä R, Suojanen J, Laine M, Erawijattari PP, Lahti L, Karihtala P, Ruuska TS, Reunanen J Journal J Adv Res Abstract Gut microbiome-derived nanoparticles, known as bacterial extracellular vesicles (bEVs), have garnere (show more...)Gut microbiome-derived nanoparticles, known as bacterial extracellular vesicles (bEVs), have garnered interest as promising tools for studying the link between the gut microbiome and human health. The diverse composition of bEVs, including their proteins, mRNAs, metabolites, and lipids, makes them useful for investigating diseases such as cancer. However, conventional approaches for studying gut microbiome composition alone may not be accurate in deciphering host-gut microbiome communication. In clinical microbiome research, there is a gap in the knowledge on the role of bEVs in solid tumor patients. (hide) EV-METRIC 50% (76th 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 Stool 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 Density gradient Filtration (Differential) (ultra)centrifugation Size-exclusion chromatography (commercial) Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) - Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Stool Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 0.2 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 100000 Filtration steps Larger than 0.45 µm/ Between 0.22 and 0.45 µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Other Name other separation method Size-exclusion chromatography (commercial) Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.52E+08 EM EM-type Transmission-EM Image type Wide-field

	EV240045	3/6	Homo sapiens	MCF7	Filtration UF Tween wash	Pauwels J	2025	50%
Study summary Full title Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics. All authors Pauwels J, Van de Steene T, Van de Velde J, De Muyer F, De Pauw D, Baeke F, Eyckerman S, Gevaert K Journal Mol Cell Proteomics Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the ex (show more...)Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 Filtration Ultrafiltration Tween wash Protein markers EV: None non-EV: bovine proteins Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 95 Cell count 400000 Separation Method Filtration steps 0.2 or 0.22µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Other Name other separation method Tween wash Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 145 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+08

	EV240164	5/5	Mesocricetus auratus	BHK-21	(d)(U)C DG	Xiong J	2025	43%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 43% (81st 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: None non-EV: None Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Mesocricetus auratus Sample Type Cell culture supernatant EV-producing cells BHK-21 EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g Cell viability (%) 80 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 Swinging bucket Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 20% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 3.5 Orientation Top-down Speed (g) 100,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240045	4/6	Homo sapiens	HEK293T	(d)(U)C Filtration	Pauwels J	2025	43%
Study summary Full title Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics. All authors Pauwels J, Van de Steene T, Van de Velde J, De Muyer F, De Pauw D, Baeke F, Eyckerman S, Gevaert K Journal Mol Cell Proteomics Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the ex (show more...)Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation. (hide) EV-METRIC 43% (81st 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 Gag-eGFP OE 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: None non-EV: bovine proteins Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 90 Wash: Rotor Type TLA-120.2 Wash: speed (g) 100000 Filtration steps 0.2 or 0.22µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240045	1/6	Homo sapiens	Blood plasma	Filtration UF	Pauwels J	2025	33%
Study summary Full title Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics. All authors Pauwels J, Van de Steene T, Van de Velde J, De Muyer F, De Pauw D, Baeke F, Eyckerman S, Gevaert K Journal Mol Cell Proteomics Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the ex (show more...)Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation. (hide) EV-METRIC 33% (64th 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Filtration steps 0.2 or 0.22µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 146 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.60E+07 EM EM-type Transmission-EM Image type Close-up

	EV240045	2/6	Homo sapiens	Urine	Filtration UF	Pauwels J	2025	33%
Study summary Full title Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics. All authors Pauwels J, Van de Steene T, Van de Velde J, De Muyer F, De Pauw D, Baeke F, Eyckerman S, Gevaert K Journal Mol Cell Proteomics Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the ex (show more...)Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward proteome analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain abundant proteins at concentrations that vastly exceed the concentrations of proteins found in the EV proteome. Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion, and precipitation methods. Here, we describe filter-aided extracellular vesicle enrichment (FAEVEr) as an approach based on 300 kDa molecular weight cutoff filtration that allows the processing of multiple samples in parallel within a reasonable time frame and at moderate cost. We demonstrate that FAEVEr is capable of quantitatively retaining EV particles on filters, while allowing extensive washing with the mild detergent Tween-20 to remove interfering non-EV proteins. The retained particles are directly lysed on the filter for a complete recovery of the EV protein cargo toward proteome analysis. Here, we validate and optimize FAEVEr on recombinant EV material and apply it on conditioned medium as well as on complex bovine serum, human plasma, and urine. Our results indicate that EVs isolated from MCF7 cells cultured with or without serum have a drastic different proteome because of nutrient deprivation. (hide) EV-METRIC 33% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration Ultrafiltration Protein markers EV: None non-EV: Albumin/ Uromodulin Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method Filtration steps 0.2 or 0.22µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240164	1/5	Homo sapiens	HeLa	(d)(U)C IAF	Xiong J	2025	13%
Study summary Full title Extracellular vesicles promote the infection and pathogenicity of Japanese encephalitis virus. All authors Xiong J, Yang L, Nan X, Zhu S, Yan M, Xiang S, Zhang L, Li Q, Yang C, Wang X, Wei N, Chen H, Si Y, Cao S, Ye J Journal J Extracell Vesicles Abstract Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to (show more...)Japanese encephalitis virus (JEV) is a neurotropic zoonotic pathogen that poses a serious threat to public health. Currently, there is no specific therapeutic agent available for JEV infection, primarily due to the complexity of its infection mechanism and pathogenesis. Extracellular vesicles (EVs) have been known to play an important role in viral infection, but their specific functions in JEV infection remain unknown. Here, ultracentrifugation in combination with density gradient centrifugation was conducted to purify EVs from JEV-infected cells. The purified EVs were found to be infectious, with virions observed inside the EVs. Furthermore, our study showed the formation process of virion-containing EVs both in vitro and in vivo, which involved the fusion of multivesicular bodies with the cell membrane, leading to the release of virion-containing intraluminal vesicles into the extracellular space. Further studies revealed that EVs played a crucial role in JEV propagation by facilitating viral entry and assembly-release. Furthermore, EVs assisted JEV in evading the neutralizing antibodies and promoted viral capability to cross the blood-brain and placental barriers. Moreover, in vivo experiments demonstrated that EVs were beneficial for JEV infection and pathogenicity. Taken together, our findings highlight the significant contribution of EVs in JEV infection and provide valuable insights into JEV pathogenesis. (hide) EV-METRIC 13% (34th 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 Japanese encephalitis virus-infected 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 Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biomarker/Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS 4h at 150,000g 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Swinging bucket Pelleting: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 250-500

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