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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190072	1/4	Homo sapiens	HUVEC	(d)(U)C IAF	Hosseinkhani, Baharak	2020	78%
Study summary Full title (Sub)populations of extracellular vesicles released by TNF-α -triggered human endothelial cells promote vascular inflammation and monocyte migration All authors Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels Journal J Extracell Vesicles Abstract Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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 TNF-treated 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 (d)(U)C IAF Protein markers EV: / ANXA2/ CD63/ CD9/ ICAM1 non-EV: BAX Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 98 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) Wash: time (min) Wash: Rotor Type Wash: speed (g) Immunoaffinity capture Selected surface protein(s) ICAM1 EV-subtype Used subtypes ICAM1 positive Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ ICAM1/ ANXA2 Not detected contaminants BAX ELISA Antibody details provided? No Detected EV-associated proteins ICAM1 Other 1 Inflammation array C3 Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190072	2/4	Homo sapiens	HUVEC	(d)(U)C IAF	Hosseinkhani, Baharak	2020	78%
Study summary Full title (Sub)populations of extracellular vesicles released by TNF-α -triggered human endothelial cells promote vascular inflammation and monocyte migration All authors Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels Journal J Extracell Vesicles Abstract Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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 TNF-treated 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 (d)(U)C IAF Protein markers EV: / ANXA2/ CD63/ CD9/ ICAM1 non-EV: BAX Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 98 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) Wash: time (min) Wash: Rotor Type Wash: speed (g) Immunoaffinity capture Selected surface protein(s) ICAM1 EV-subtype Used subtypes ICAM1 negative Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Not detected EV-associated proteins CD9/ CD63/ ICAM1/ ANXA2 Not detected contaminants BAX ELISA Antibody details provided? No Not detected EV-associated proteins Not detected contaminants ICAM1 Other 1 Inflammation array C3 Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190072	3/4	Homo sapiens	HUVEC	(d)(U)C IAF	Hosseinkhani, Baharak	2020	78%
Study summary Full title (Sub)populations of extracellular vesicles released by TNF-α -triggered human endothelial cells promote vascular inflammation and monocyte migration All authors Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels Journal J Extracell Vesicles Abstract Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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 TNF-treated 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 (d)(U)C IAF Protein markers EV: / ANXA2/ CD63/ CD9/ ICAM1 non-EV: BAX Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 98 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type S-4-72 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) Wash: time (min) Wash: Rotor Type Wash: speed (g) Immunoaffinity capture Selected surface protein(s) ICAM1 EV-subtype Used subtypes ICAM1 positive Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ ANXA2/ ICAM1 Not detected EV-associated proteins Not detected contaminants BAX ELISA Antibody details provided? No Detected EV-associated proteins ICAM1 Other 1 Inflammation array C3 Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-400 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190072	4/4	Homo sapiens	HUVEC	(d)(U)C IAF	Hosseinkhani, Baharak	2020	78%
Study summary Full title (Sub)populations of extracellular vesicles released by TNF-α -triggered human endothelial cells promote vascular inflammation and monocyte migration All authors Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels Journal J Extracell Vesicles Abstract Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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 TNF-treated 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 (d)(U)C IAF Protein markers EV: / ANXA2/ CD63/ CD9/ ICAM1 non-EV: BAX Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 98 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type S-4-72 Pelleting: speed (g) 10000 Immunoaffinity capture Selected surface protein(s) ICAM1 EV-subtype Used subtypes ICAM1 negative Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ ANXA2 Not detected EV-associated proteins ICAM1 Not detected contaminants BAX ELISA Antibody details provided? No Not detected EV-associated proteins Not detected contaminants ICAM1 Other 1 Inflammation array C3 Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-400 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV190072
species	Homo sapiens
sample type	Cell culture
cell type	HUVEC
condition	TNF-treated
separation protocol	(d)(U)C IAF	(d)(U)C IAF	(d)(U)C IAF	(d)(U)C IAF
EV subtype	ICAM1 positive	ICAM1 negative	ICAM1 positive	ICAM1 negative
Exp. nr.	1	2	3	4
EV-METRIC %	78	78	78	78