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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220279	1/5	Homo sapiens	Umbilical cord blood-mesenchymal stem cells	(d)(U)C	Sung DK	2019	44%
Study summary Full title Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content. All authors Sung DK, Chang YS, Sung SI, Ahn SY, Park WS Journal J Clin Med Abstract The aim of this study was to determine the optimal preconditioning regimen for the wound healing the (show more...)The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), HO (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via ERK1/2 and AKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content. (hide) EV-METRIC 44% (84th 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 Protein markers EV: CD81/ CD63/ CD9/ Angiogenin/ Angiopoietin-1/ HGF/ IGFBP-2/ IGFBP-3/ Pentraxin-3/ Serpin E1/ TIMP-1/ Thrombospondin-1/ uPA/ VEGF non-EV: Cytochrome C/ Fibrillarin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord blood-mesenchymal stem cells EV-harvesting Medium Serum free medium Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000rp Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Fibrillarin/ Cytochrome C/ GM130 ELISA Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Angiogenin/ Angiopoietin-1/ HGF/ VEGF Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 30-100 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Scanning-EM Image type Close-up

	EV220279	2/5	Homo sapiens	Umbilical cord blood-mesenchymal stem cells	(d)(U)C	Sung DK	2019	44%
Study summary Full title Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content. All authors Sung DK, Chang YS, Sung SI, Ahn SY, Park WS Journal J Clin Med Abstract The aim of this study was to determine the optimal preconditioning regimen for the wound healing the (show more...)The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), HO (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via ERK1/2 and AKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content. (hide) EV-METRIC 44% (84th 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 Thrombin 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 Protein markers EV: CD81/ CD63/ CD9/ Angiogenin/ Angiopoietin-1/ HGF/ IGFBP-2/ IGFBP-3/ Pentraxin-3/ Serpin E1/ TIMP-1/ Thrombospondin-1/ uPA/ VEGF non-EV: Cytochrome C/ Fibrillarin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord blood-mesenchymal stem cells EV-harvesting Medium Serum free medium Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000rp Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Fibrillarin/ Cytochrome C/ GM130 ELISA Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Angiogenin/ Angiopoietin-1/ HGF/ VEGF Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 30-100 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Scanning-EM Image type Close-up

	EV220279	3/5	Homo sapiens	Umbilical cord blood-mesenchymal stem cells	(d)(U)C	Sung DK	2019	44%
Study summary Full title Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content. All authors Sung DK, Chang YS, Sung SI, Ahn SY, Park WS Journal J Clin Med Abstract The aim of this study was to determine the optimal preconditioning regimen for the wound healing the (show more...)The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), HO (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via ERK1/2 and AKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin LPS Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD81/ CD63/ CD9/ Angiogenin/ Angiopoietin-1/ HGF/ IGFBP-2/ IGFBP-3/ Pentraxin-3/ Serpin E1/ TIMP-1/ Thrombospondin-1/ uPA/ VEGF non-EV: Cytochrome C/ Fibrillarin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord blood-mesenchymal stem cells EV-harvesting Medium Serum free medium Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000rp Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Fibrillarin/ Cytochrome C/ GM130 ELISA Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Angiogenin/ Angiopoietin-1/ HGF/ VEGF Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 30-100 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Scanning-EM Image type Close-up

	EV220279	4/5	Homo sapiens	Umbilical cord blood-mesenchymal stem cells	(d)(U)C	Sung DK	2019	44%
Study summary Full title Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content. All authors Sung DK, Chang YS, Sung SI, Ahn SY, Park WS Journal J Clin Med Abstract The aim of this study was to determine the optimal preconditioning regimen for the wound healing the (show more...)The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), HO (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via ERK1/2 and AKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content. (hide) EV-METRIC 44% (84th 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 H2O2 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 Protein markers EV: CD81/ CD63/ CD9/ Angiogenin/ Angiopoietin-1/ HGF/ IGFBP-2/ IGFBP-3/ Pentraxin-3/ Serpin E1/ TIMP-1/ Thrombospondin-1/ uPA/ VEGF non-EV: Cytochrome C/ Fibrillarin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord blood-mesenchymal stem cells EV-harvesting Medium Serum free medium Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000rp Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants Cytochrome C Not detected contaminants Fibrillarin/ GM130 ELISA Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Angiogenin/ Angiopoietin-1/ HGF/ VEGF Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 30-100 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Scanning-EM Image type Close-up

	EV220279	5/5	Homo sapiens	Umbilical cord blood-mesenchymal stem cells	(d)(U)C	Sung DK	2019	44%
Study summary Full title Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content. All authors Sung DK, Chang YS, Sung SI, Ahn SY, Park WS Journal J Clin Med Abstract The aim of this study was to determine the optimal preconditioning regimen for the wound healing the (show more...)The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), HO (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via ERK1/2 and AKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content. (hide) EV-METRIC 44% (84th 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 Hypoxia 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 Protein markers EV: CD81/ CD63/ CD9/ Angiogenin/ Angiopoietin-1/ HGF/ IGFBP-2/ IGFBP-3/ Pentraxin-3/ Serpin E1/ TIMP-1/ Thrombospondin-1/ uPA/ VEGF non-EV: Cytochrome C/ Fibrillarin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord blood-mesenchymal stem cells EV-harvesting Medium Serum free medium Cell count Not reported Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000rp Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants Cytochrome C Not detected contaminants Fibrillarin/ GM130 ELISA Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Angiogenin/ Angiopoietin-1/ HGF/ VEGF Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 30-100 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Scanning-EM Image type Close-up

	EV220196	1/4	Bos taurus	BL20 (immortalised bovine lymphosarcoma)	(d)(U)C	Gillan V	2019	44%
Study summary Full title Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes. All authors Gillan V, Simpson DM, Kinnaird J, Maitland K, Shiels B, Devaney E Journal Cell Microbiol Abstract The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular euka (show more...)The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation-like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection-associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta-miR-181a and bta-miR-181b, we identified putative messenger RNA targets and confirmed the interaction of bta-miR181a with ICAM-1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection. (hide) EV-METRIC 44% (84th 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 Protein markers EV: Rab-5B/ CD63 non-EV: Cyc1 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells BL20 (immortalised bovine lymphosarcoma) EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Surespin 630 (17 ml) Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 70 Wash: Rotor Type Surespin 630 (17 ml) Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Rab-5B Not detected contaminants Cyc1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up Report size (nm) 100 Other particle analysis name(1) No

	EV220196	3/4	Bos taurus	BL20 (immortalised bovine lymphosarcoma)	(d)(U)C	Gillan V	2019	44%
Study summary Full title Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes. All authors Gillan V, Simpson DM, Kinnaird J, Maitland K, Shiels B, Devaney E Journal Cell Microbiol Abstract The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular euka (show more...)The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation-like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection-associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta-miR-181a and bta-miR-181b, we identified putative messenger RNA targets and confirmed the interaction of bta-miR181a with ICAM-1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection. (hide) EV-METRIC 44% (84th 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 Theileria annulata infected (TBL20) 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 Protein markers EV: Rab-5B/ CD63 non-EV: Cyc1 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells BL20 (immortalised bovine lymphosarcoma) EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Surespin 630 (17 ml) Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 70 Wash: Rotor Type Surespin 630 (17 ml) Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Rab-5B Not detected contaminants Cyc1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up Report size (nm) 100 Other particle analysis name(1) No

	EV210038	1/1	Homo sapiens	NCI-H460	(d)(U)C Filtration	Wang, Shuang	2019	44%
Study summary Full title Autocrine secretions enhance radioresistance in an exosome‑independent manner in NSCLC cells All authors Shuang Wang, Piaoyang Gao, Na Li, Ping Chen, Jinhan Wang, Ningning He, Kaihua Ji, Liqing Du, Qiang Liu Journal Int J Oncol Abstract Radiotherapy resistance in patient with non‑small cell lung cancer (NSCLC) reduces patient surviva (show more...)Radiotherapy resistance in patient with non‑small cell lung cancer (NSCLC) reduces patient survival and remains a significant challenge for the treatment of NSCLC. Radiation resistance has been demonstrated to be affected by secreted factors, yet it remains unclear how autocrine secretions affect the radioresistance of NSCLC cells. In the present study, the NSCLC cell line, NCI‑H460, was irradiated with γ‑rays (4 Gy) and then cultured in medium from H460 cells or normal medium to examine the potential influence of cell secretions on the radiation resistance of H460 cells. Cell viability, accumulation of reactive oxygen species and DNA repair capacity were all markedly improved in the irradiated H460 cells that were cultured in conditioned medium (CM), compared with those cells cultured in normal medium. In addition, G2/M cell cycle arrest and upregulation of homologous recombination repair proteins were observed in the CM‑treated cells, while exosomes secreted by H460 cells had no influence on the radiation resistance of H460 cells. Taken together, these results indicate that autocrine secretions enhance the radiation resistance of γ‑irradiated H460 cells and that these secretions mainly affect the DNA repair process. (hide) EV-METRIC 44% (84th 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 Filtration Protein markers EV: TSG101/ B-actin/ CD9/ B-tubulin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NCI-H460 EV-harvesting Medium Serum free medium 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) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected EV-associated proteins B-actin/ B-tubulin/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV200148	1/4	Homo sapiens	Blood plasma	"(d)(U)C Filtration SEC (non-commercial) UF"	Menon, Ramkumar	2019	44%
Study summary Full title Quantitative Proteomics by SWATH-MS of Maternal Plasma Exosomes Determine Pathways Associated With Term and Preterm Birth All authors Ramkumar Menon, Christopher Luke Dixon, Samantha Sheller-Miller, Stephen J Fortunato, George R Saade, Carlos Palma, Andrew Lai, Dominic Guanzon, Carlos Salomon Journal Endocrinology Abstract Exosomes are membrane-bound nanovesicles that transport molecular signals between cells. This study (show more...)Exosomes are membrane-bound nanovesicles that transport molecular signals between cells. This study determined changes in maternal plasma exosome proteomics contents in term and preterm births. Maternal plasma (MP) samples were collected from group 1: term not in labor (TNIL, n = 13); group 2: term in labor (TL, n = 11); group 3: preterm premature rupture of membranes (pPROM, n = 8); and group 4: preterm birth (PTB, n = 13). Exosomes isolated from plasma by differential density centrifugation followed by size exclusion chromatography were characterized by morphology (electron microscopy), quantity and size (nanoparticle tracking analysis), and markers (western blot). A quantitative, information-independent acquisition [sequential windowed acquisition of all theoretical mass spectra (SWATH-MS)] approach was used to determine the protein profile in exosomes. Ingenuity Pathway Analysis determined pathways associated with the protein profile identified in exosomes. MP exosomes were spherical, had a mean diameter of 120 nm, and were positive for exosomal proteins CD63 and TSG101 irrespective of pregnancy status. No distinct changes in exosome quantities were seen in maternal circulation across the groups. SWATH-MS identified 72 statistically significant proteins across the groups studied. Bioinformatics analysis showed the proteins within the exosomes in TNIL, TL, pPROM, and PTB target pathways mainly associated with inflammatory and metabolic signals. Exosomal data suggest that homeostatic imbalances, specifically inflammatory and endocrine signaling, might disrupt pregnancy maintenance resulting in labor-related changes both at term and preterm. Reflection of physiologic changes in exosomes is suggestive of its usefulness as biomarkers and cellular function indicators. (hide) EV-METRIC 44% (77th 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 "Pregnant; Term, not in labor" 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 Filtration Size exclusion chromatography (non-commercial) Ultrafiltration" Protein markers EV: "TSG101/ PLAP/ CD63/ CD9" non-EV: Grp94 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Size-exclusion chromatography Total column volume (mL) Not specified Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Other Name other separation method "(Differential) (ultra)centrifugation Other Name other separation method Size exclusion chromatography (non-commercial) Other Name other separation method Ultrafiltration" Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins "CD9/ CD63/ TSG101" Not detected contaminants Grp94 Proteomics database No Fluorescent NTA Antibody details provided? No Detected EV-associated proteins "PLAP/ CD63" Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 134 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV190092	2/2	Homo sapiens	HT1080	(d)(U)C	Brassart B	2019	44%
Study summary Full title Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA. All authors Brassart B, Da Silva J, Donet M, Seurat E, Hague F, Terryn C, Velard F, Michel J, Ouadid-Ahidouch H, Monboisse JC, Hinek A, Maquart FX, Ramont L, Brassart-Pasco S. Journal Br J Cancer Abstract BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elasto (show more...)BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases. METHODS: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA. RESULTS: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding. CONCLUSIONS: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites. (hide) EV-METRIC 44% (84th 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 elastin degradation product-stimulated 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 Protein markers EV: CD9/ CD81/ RhoA/ HSP90/ Actin/ Integrin-alphaV/ P-ERM/ MMP-2/ MMP-14 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT1080 EV-harvesting Medium Serum free medium Cell viability (%) 94 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type JLA110 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Wash: time (min) Wash: Rotor Type Wash: speed (g) Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD81/ RhoA/ HSP90/ Actin/ Integrin-alphaV/ P-ERM/ MMP-2/ MMP-14 Not detected contaminants GM130 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins Not detected contaminants Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type EV concentration EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190019	1/4	Mus musculus	C2C12	DG (d)(U)C	Leermakers PA	2019	44%
Study summary Full title Iron deficiency-induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria-containing vesicles. All authors Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR. Journal FASEB J Abstract Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: HSP70/ Flotillin1 non-EV: Proteomics no EV density (g/ml) 1.12-1.19 g/ml Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells C2C12 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: time(min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 188000 Duration (min) 16 Fraction volume (mL) 1 ml Fraction processing Centrifugation Pelleting: volume per fraction 6 ml Pelleting: duration (min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes sedimentation speed;Other Used subtypes 16,000g Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSP70 Characterization: Lipid analysis No EM EM-type Cryo-EM Image type Wide-field

	EV190019	2/4	Mus musculus	C2C12	DG (d)(U)C	Leermakers PA	2019	44%
Study summary Full title Iron deficiency-induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria-containing vesicles. All authors Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR. Journal FASEB J Abstract Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: HSP70/ Flotillin1 non-EV: Proteomics no EV density (g/ml) 1.12-1.19 g/ml Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells C2C12 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: time(min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 188000 Duration (min) 16 Fraction volume (mL) 1 ml Fraction processing Centrifugation Pelleting: volume per fraction 6 ml Pelleting: duration (min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes sedimentation speed;Other Used subtypes 100,000g Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSP70 Characterization: Lipid analysis No EM EM-type Cryo-EM Image type Wide-field

	EV190019	3/4	Mus musculus	C2C12	DG (d)(U)C	Leermakers PA	2019	44%
Study summary Full title Iron deficiency-induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria-containing vesicles. All authors Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR. Journal FASEB J Abstract Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide) EV-METRIC 44% (84th 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 Deferiprone/iron chelation Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: HSP70/ Flotillin1 non-EV: Proteomics no EV density (g/ml) 1.12-1.19 g/ml Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells C2C12 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: time(min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 188000 Duration (min) 16 Fraction volume (mL) 1 ml Fraction processing Centrifugation Pelleting: volume per fraction 6 ml Pelleting: duration (min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes sedimentation speed;Other Used subtypes 16,000g Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSP70 Characterization: Lipid analysis No EM EM-type Cryo-EM Image type Wide-field

	EV190019	4/4	Mus musculus	C2C12	DG (d)(U)C	Leermakers PA	2019	44%
Study summary Full title Iron deficiency-induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria-containing vesicles. All authors Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR. Journal FASEB J Abstract Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide) EV-METRIC 44% (84th 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 Deferiprone/iron chelation Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: HSP70/ Flotillin1 non-EV: Proteomics no EV density (g/ml) 1.12-1.19 g/ml Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells C2C12 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: time(min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 188000 Duration (min) 16 Fraction volume (mL) 1 ml Fraction processing Centrifugation Pelleting: volume per fraction 6 ml Pelleting: duration (min) 65 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes sedimentation speed;Other Used subtypes 100,000g Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSP70 Characterization: Lipid analysis No EM EM-type Cryo-EM Image type Wide-field

	EV190016	1/1	Homo sapiens	Wharton's jelly mesenchymal stromal cells	(d)(U)C	Thomi, Gierin	2019	44%
Study summary Full title Exosomes derived from umbilical cord mesenchymal stem cells reduce microglia-mediated neuroinflammation in perinatal brain injury All authors Gierin Thomi, Daniel Surbek, Valérie Haesler, Marianne Joerger-Messerli, Andreina Schoeberlein Journal Stem Cell Res Ther Abstract Background: Preterm newborns are at high risk of developing neurodevelopmental deficits caused by ne (show more...)Background: Preterm newborns are at high risk of developing neurodevelopmental deficits caused by neuroinflammation leading to perinatal brain injury. Human Wharton's jelly mesenchymal stem cells (hWJ-MSC) derived from the umbilical cord have been suggested to reduce neuroinflammation, in part through the release of extracellular vesicle-like exosomes. Here, we studied whether exosomes derived from hWJ-MSC have anti-inflammatory effects on microglia-mediated neuroinflammation in perinatal brain injury. Methods: Using ultracentrifugation, we isolated exosomes from hWJ-MSC culture supernatants. In an in vitro model of neuroinflammation, we stimulated immortalized BV-2 microglia and primary mixed glial cells with lipopolysaccharide (LPS) in the presence or absence of exosomes. In vivo, we introduced brain damage in 3-day-old rat pups and treated them intranasally with hWJ-MSC-derived exosomes. Results: hWJ-MSC-derived exosomes dampened the LPS-induced expression of inflammation-related genes by BV-2 microglia and primary mixed glial cells. The secretion of pro-inflammatory cytokines by LPS-stimulated primary mixed glial was inhibited by exosomes as well. Exosomes interfered within the Toll-like receptor 4 signaling of BV-2 microglia, as they prevented the degradation of the NFκB inhibitor IκBα and the phosphorylation of molecules of the mitogen-activated protein kinase family in response to LPS stimulation. Finally, intranasally administered exosomes reached the brain and reduced microglia-mediated neuroinflammation in rats with perinatal brain injury. Conclusions: Our data suggest that the administration of hWJ-MSC-derived exosomes represents a promising therapy to prevent and treat perinatal brain injury. (hide) EV-METRIC 44% (84th 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ ICAM/ Flotillin1/ EpCAM/ ANXA5 non-EV: GRP94/ GM130 Proteomics no Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Wharton's jelly mesenchymal stromal cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type TFT-70 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 70 Wash: Rotor Type TFT-70 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? No Not detected contaminants GRP94 Detected EV-associated proteins Alix/ Flotillin1/ CD63/ EpCAM/ ICAM/ ANXA5/ CD81/ TSG101 Not detected contaminants GM130 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field Report size (nm) 42.93

	EV190011	2/5	Mus musculus	E0771	(d)(U)C	Cianciaruso C	2019	44%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 44% (84th 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 (d)(U)C Protein markers EV: CD81/ Alix/ CD9/ GAPDH non-EV: Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells E0771 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ GAPDH Not detected EV-associated proteins CD81 Characterization: Lipid analysis No

	EV170031	1/1	Homo sapiens	Serum	(d)(U)C Filtration	Ramanathan S	2019	44%
Study summary Full title Exosome microRNA signatures in patients with complex regional pain syndrome undergoing plasma exchange. All authors Ramanathan S, Douglas SR, Alexander GM, Shenoda BB, Barrett JE, Aradillas E, Sacan A, Ajit SK Journal J Transl Med Abstract BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employ (show more...)BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE. (hide) EV-METRIC 44% (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 Serum Sample origin Complex regional pain syndrome 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 (d)(U)C Filtration Adj. k-factor 142.9 (pelleting) / 142.9 (washing) Protein markers EV: CD81/ CD63 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 142.9 Wash: time (min) 70 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 142.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 85.7 EV concentration Yes Particle yield 394000000000 EM EM-type Transmission-EM/ Immune-EM EM protein CD81 Image type Close-up, Wide-field

	EV230047	3/4	Homo sapiens	Serum	(d)(U)C Filtration UF	Panigrahi GK	2019	43%
Study summary Full title Exosome proteomic analyses identify inflammatory phenotype and novel biomarkers in African American prostate cancer patients. All authors Panigrahi GK, Praharaj PP, Kittaka H, Mridha AR, Black OM, Singh R, Mercer R, van Bokhoven A, Torkko KC, Agarwal C, Agarwal R, Abd Elmageed ZY, Yadav H, Mishra SK, Deep G Journal Cancer Med Abstract African American men face a stark prostate cancer (PCa)-related health disparity, with the highest i (show more...)African American men face a stark prostate cancer (PCa)-related health disparity, with the highest incidence and mortality rates compared to other races. Additional and innovative measures are warranted to reduce this health disparity. Here, we focused on the identification of a novel serum exosome-based "protein signature" for potential use in the early detection and better prognosis of PCa in African American men. Nanoparticle tracking analyses showed that compared to healthy individuals, exosome concentration (number/ml) was increased by ~3.2-fold (P ˂ 0.05) in the sera of African American men with PCa. Mass spectrometry-based proteomic analysis of serum exosomes identified seven unique and fifty-five overlapping proteins (up- or downregulated) in African Americans with PCa compared to healthy African Americans. Furthermore, ingenuity pathway analyses identified the inflammatory acute-phase response signaling as the top pathway associated with proteins loaded in exosomes from African American PCa patients. Interestingly, African American PCa E006AA-hT cells secreted exosomes strongly induced a proinflammatory M2-phenotype in macrophages and showed calcium response on sensory neurons, suggesting a neuroinflammatory response. Additionally, proteomic analyses showed that the protein Isoform 2 of Filamin A has higher loading (2.6-fold) in exosomes from African Americans with PCa, but a lesser loading (0.6-fold) was observed in exosomes from Caucasian men with PCa compared to race-matched healthy individuals. Interestingly, TCGA and Taylor's dataset as well as IHC analyses of PCa tissue showed a lower Filamin A expression in tissues of PCa patients compared with normal subjects. Overall, these results support the usefulness of serum exosomes to noninvasively detect inflammatory phenotype and to discover novel biomarkers associated with PCa in African American men. (hide) EV-METRIC 43% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70.1 Ti Pelleting: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 150 Membrane type NS Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 180 EV concentration Yes

	EV230047	4/4	Homo sapiens	Serum	(d)(U)C Filtration UF	Panigrahi GK	2019	43%
Study summary Full title Exosome proteomic analyses identify inflammatory phenotype and novel biomarkers in African American prostate cancer patients. All authors Panigrahi GK, Praharaj PP, Kittaka H, Mridha AR, Black OM, Singh R, Mercer R, van Bokhoven A, Torkko KC, Agarwal C, Agarwal R, Abd Elmageed ZY, Yadav H, Mishra SK, Deep G Journal Cancer Med Abstract African American men face a stark prostate cancer (PCa)-related health disparity, with the highest i (show more...)African American men face a stark prostate cancer (PCa)-related health disparity, with the highest incidence and mortality rates compared to other races. Additional and innovative measures are warranted to reduce this health disparity. Here, we focused on the identification of a novel serum exosome-based "protein signature" for potential use in the early detection and better prognosis of PCa in African American men. Nanoparticle tracking analyses showed that compared to healthy individuals, exosome concentration (number/ml) was increased by ~3.2-fold (P ˂ 0.05) in the sera of African American men with PCa. Mass spectrometry-based proteomic analysis of serum exosomes identified seven unique and fifty-five overlapping proteins (up- or downregulated) in African Americans with PCa compared to healthy African Americans. Furthermore, ingenuity pathway analyses identified the inflammatory acute-phase response signaling as the top pathway associated with proteins loaded in exosomes from African American PCa patients. Interestingly, African American PCa E006AA-hT cells secreted exosomes strongly induced a proinflammatory M2-phenotype in macrophages and showed calcium response on sensory neurons, suggesting a neuroinflammatory response. Additionally, proteomic analyses showed that the protein Isoform 2 of Filamin A has higher loading (2.6-fold) in exosomes from African Americans with PCa, but a lesser loading (0.6-fold) was observed in exosomes from Caucasian men with PCa compared to race-matched healthy individuals. Interestingly, TCGA and Taylor's dataset as well as IHC analyses of PCa tissue showed a lower Filamin A expression in tissues of PCa patients compared with normal subjects. Overall, these results support the usefulness of serum exosomes to noninvasively detect inflammatory phenotype and to discover novel biomarkers associated with PCa in African American men. (hide) EV-METRIC 43% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Prostate cancer 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70.1 Ti Pelleting: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 150 Membrane type NS Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 170 EV concentration Yes

	EV210430	1/2	Homo sapiens	Bone-marrow derived mesenchymal stem cells	Total Exosome Isolation	Tracy SA	2019	43%
Study summary Full title A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid. All authors Tracy SA, Ahmed A, Tigges JC, Ericsson M, Pal AK, Zurakowski D, Fauza DO Journal J Pediatr Surg Abstract Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapi (show more...)Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases. (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 Commercial method Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Bone-marrow derived mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Antibody details provided? Yes Antibody dilution provided? No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 151 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field

	EV210430	2/2	Homo sapiens	Amniotic fluid derived mesenchymal stem cells	Total Exosome Isolation	Tracy SA	2019	43%
Study summary Full title A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid. All authors Tracy SA, Ahmed A, Tigges JC, Ericsson M, Pal AK, Zurakowski D, Fauza DO Journal J Pediatr Surg Abstract Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapi (show more...)Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases. (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 Commercial method Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Amniotic fluid derived mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Antibody details provided? Yes Antibody dilution provided? No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 132 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field

	EV190020	3/3	Rattus norvegicus	ASML	DG (d)(U)C Filtration	Kyuno, Daisuke	2019	43%
Study summary Full title Claudin7-dependent exosome-promoted reprogramming of non-metastasizing tumor cells. All authors Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M. Journal Int J Cancer Abstract Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (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 claudin 7 knockdown 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 DG (d)(U)C Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.15-1.56 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells ASML EV-harvesting Medium Serum free medium Cell viability (%) 100 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) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1.28 Fraction processing Centrifugation Pelleting: volume per fraction 50 Pelleting: duration (min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 50 Pelleting-wash: duration (min) 150 Pelleting-wash: speed (g) Type 45 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: RNA analysis RNA analysis Type Database Proteinase treatment RNAse treatment Characterization: Lipid analysis No

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