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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210360	2/2	Homo sapiens	Urine	DG (d)(U)C	Abe H	2018	33%
Study summary Full title Urinary Exosomal mRNA of WT1 as Diagnostic and Prognostic Biomarker for Diabetic Nephropathy. All authors Abe H, Sakurai A, Ono H, Hayashi S, Yoshimoto S, Ochi A, Ueda S, Nishimura K, Shibata E, Tamaki M, Kishi F, Kishi S, Murakami T, Nagai K, Doi T Journal J Med Invest Abstract Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with incre (show more...)Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality as compared to other causes of renal disease. Albuminuria is often the first clinical indicator of the presence of DN. However, albuminuria or proteinuria is a common symptom in patients with various renal disorders. Therefore, specific biomarkers for the diagnosis of DN are required. A primary hallmark of DN is the progressive damage and death of glomerular podocytes, resulting in the leaking of proteins into the urine. Urinary exosomes released by podocytes are microvesicles containing information of the originated cells. Podocyte-derived signal transduction factors (PDSTFs) are good candidates to assess podocyte injuries. The profile of PDSTFs in urinary exosomes from patients with DN is different from that from patients with minimal change nehrotic syndrome. In addition, PDSTFs molecules in exosomes were derived from primary murine podocytes under high glucose conditions. Among PDSTFs in urinary exosomes, Wilms tumor 1 (WT1) levels reflected damage of diabetic glomeruli in the patients. Urinary exosomal WT1 can predict the decline in eGFR for the following several years. In conclusion, urinary exosomal WT1 is a useful biomarker to improve risk stratification in patients with DN. J. Med. Invest. 65:208-215, August, 2018. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Proteinuria patients 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 Density gradient (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type RP-65 Pelleting: speed (g) 70000 Density gradient Type Continuous Lowest density fraction 0.25 M Highest density fraction 2 M Total gradient volume, incl. sample (mL) 30 Sample volume (mL) 5 Orientation Top-down Rotor type RPS-28SA Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 60 Pelleting: rotor type RPS-50-2 Pelleting: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV210349	1/13	Homo sapiens	NA	(d)(U)C	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (61st 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 NA 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: TSG101/ CD63/ CD81/ Syntenin/ CD9/ CHMP1A/ SHH non-EV: gp96/ actin Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A 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 Pelleting performed Yes Pelleting: speed (g) 2000 Wash: volume per pellet (ml) 5 Wash: time (min) 20 Wash: Rotor Type SW 55 Ti Wash: speed (g) 2000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins SHH Not detected EV-associated proteins CD81/ Syntenin/ TSG101/ CD63/ CD9/ CHMP1A Not detected contaminants actin/ gp96 Characterization: Lipid analysis No

	EV210349	4/13	Homo sapiens	NA	(d)(U)C IAF	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (61st 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 NA 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 Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ SHH/ Syntenin/ CD81/ RAB18/ AXL/ TMED10 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) SHH Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins SHH/ CD81/ RAB18/ AXL/ TMED10 Not detected EV-associated proteins CD63/ Synthenin Characterization: Lipid analysis No

	EV210349	5/13	Homo sapiens	NA	(d)(U)C IAF	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (61st 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 NA Sample origin CHMP1A null Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ SHH/ Syntenin/ CD81/ RAB18/ AXL/ TMED10 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ SHH/ Syntenin/ CD81/ RAB18 Not detected EV-associated proteins AXL/ TMED10 Characterization: Lipid analysis No

	EV210349	6/13	Homo sapiens	NA	(d)(U)C IAF	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (61st 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 NA Sample origin CHMP1A null Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Immunoaffinity capture (non-commercial) Protein markers EV: CD9/ CD63/ SHH non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) CD9 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ SHH Characterization: Lipid analysis No

	EV210349	7/13	Homo sapiens	NA	(d)(U)C IAF	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (61st 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 NA Sample origin CHMP1A null Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ SHH/ Syntenin/ CD81/ RAB18/ AXL/ TMED10 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) IgG Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81 Not detected EV-associated proteins SHH/ Synthenin/ CD81/ RAB18/ AXL/ TMED10 Characterization: Lipid analysis No

	EV210349	8/13	Homo sapiens	NA	(d)(U)C	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (61st 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 NA Sample origin CHMP1A null 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/ Syntenin/ TSG101/ CD63/ CD9/ CHMP1A/ SHH non-EV: gp96/ actin Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A 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 Pelleting performed Yes Pelleting: speed (g) 2000 Wash: volume per pellet (ml) 5 Wash: time (min) 20 Wash: Rotor Type SW 55 Ti Wash: speed (g) 2000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin/ CD9/ CD63/ CD81/ SHH Not detected EV-associated proteins CHMP1A Characterization: Lipid analysis No

	EV210349	11/13	Homo sapiens	CSF	(d)(U)C IAF	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (78th 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 CSF 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 Immunoaffinity capture (non-commercial) Protein markers EV: SHH/ RAB18/ AXL/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type CSF 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) IgG Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected EV-associated proteins SHH/ RAB18/ AXL Characterization: Lipid analysis No

	EV210349	12/13	Homo sapiens	CSF	(d)(U)C IAF	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (78th 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 CSF 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 Immunoaffinity capture (non-commercial) Protein markers EV: SHH/ RAB18/ AXL/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type CSF 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) SHH Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ SHH/ RAB18/ AXL Characterization: Lipid analysis No

	EV210349	13/13	Homo sapiens	CSF	(d)(U)C IAF	Coulter ME	2018	33%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 33% (78th 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 CSF 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 Immunoaffinity capture (non-commercial) Protein markers EV: SHH/ RAB18/ AXL/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type CSF 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected EV-associated proteins SHH/ RAB18/ AXL Characterization: Lipid analysis No

	EV210277	2/4	Homo sapiens	Urine	(d)(U)C	Shin H	2018	33%
Study summary Full title Aqueous two-phase system to isolate extracellular vesicles from urine for prostate cancer diagnosis. All authors Shin H, Park YH, Kim YG, Lee JY, Park J Journal PLoS One Abstract Analyzing extracellular vesicles (EVs) is an attractive approach to diagnosis of prostate diagnosis. (show more...)Analyzing extracellular vesicles (EVs) is an attractive approach to diagnosis of prostate diagnosis. However, existing methods of EVs isolation have low efficiency, purity, and long process time, and therefore have low diagnostic ability. To solve these the problems, a two-phase system is adapted to isolate EVs from a patient's urine. Urine from 20 prostate cancer (PCA) patients and 10 benign prostate hyperplasia patients was used to quantify the EVs-isolation ability of an aqueous two-phase system (ATPS) and to compare the diagnostic ability of ATPS with that of the conventional diagnosis method. An optimized ATPS isolates EVs with ~100% efficiency within ~30 min, with 14 times as high as achieved by ultracentrifugation. Afterward, PCR and ELISA are used to detect EVs derived from PCA cells in urine. The results demonstrate that diagnostic ability based on ATPS is better than other conventional diagnostic methods. ATPS can obtain a high quality and quantity of EVs from patients' urine. EVs contain cancer-related protein and genes, so these abundant sources enable diagnosis with high specificity and sensitivity. Therefore, ATPS is a useful tool to increase the specificity and sensitivity of diagnosis. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Prostate cancer 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 non-EV: None Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods/New methodological development Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 4 Wash: time (min) 120 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Not detected EV-associated proteins CD9/ CD63/ CD81 ELISA Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-400 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV210172	3/6	Mus musculus	AML12	(d)(U)C	Chen, Li	2018	33%
Study summary Full title Pathways of production and delivery of hepatocyte exosomes All authors Li Chen, Ruju Chen, Sherri Kemper, David R Brigstock Journal J Cell Commun Signal Abstract Hepatocyte exosomes (ExoHep) are proposed to mediate physiological or pathophysiological signaling i (show more...)Hepatocyte exosomes (ExoHep) are proposed to mediate physiological or pathophysiological signaling in a variety of hepatic target cells. ExoHep were purified from the medium of primary mouse hepatocytes or AML12 cells and characterized as ~100 nm nanovesicles that were positive for proteins commonly found in exosomes (CD9, CD81, flotillin) or hepatocytes (asialoglycoprotein receptor). Ethanol treatment of hepatocytes caused increased ExoHep release and increased cellular mRNA expression of components involved in intracellular vesicle trafficking (Rab 5a,b,c, Rab 7a, Rab 27a,b) or exosome biogenesis via the ESCRT (HGS, Alix, STAM1, TSG101, VTA1, YKT6) or ceramide (nSmase2) pathways. RNA interference of HGS, Alix, TSG101 or nSmase 2 caused exosome production by normal or ethanol-treated hepatocytes to be reduced. In mice, in vivo administration of fluorescently-labeled ExoHep resulted in their accumulation in the liver and preferential localization to hepatic stellate cells (HSC) or hepatocytes, the latter of which showed enhanced ExoHep binding when isolated from fibrotic mice. In cell co-cultures, the intercellular transfer of RNA from hepatocytes to hepatocytes or HSC was blocked by the exosome inhibitor GW4869. ExoHep binding to HSC or hepatocytes occurred via mechanisms that involved heparin-like molecules and cellular integrin αv or β1 subunits , and resulted in a reversal of fibrosis-associated gene expression in HSC and of ethanol-induced damage in hepatocytes. These studies provide insight regarding the regulation and/or participation of exosome biogenesis or trafficking components in hepatocytes and show that ExoHep can mediate therapeutic changes in activated HSC or injured hepatocytes that occur downstream of heparin- or integrin-dependent binding interactions. (hide) EV-METRIC 33% (75th 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: ASGPR1/ CD81/ Flotillin1/ CD9 non-EV: None Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells AML12 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD81 Flow cytometry Antibody details provided? No Detected EV-associated proteins ASGPR1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 110 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210072	2/5	Homo sapiens	Well5	(d)(U)C	Gong, Liangzhi	2018	33%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 33% (75th 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: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Well5 EV-harvesting Medium Not specified 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 Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Alix/ CD81 Not detected contaminants Calnexin 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 NTA Report type Size range/distribution Reported size (nm) 1-300

	EV210072	3/5	Homo sapiens	143B	(d)(U)C	Gong, Liangzhi	2018	33%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 33% (75th 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: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 143B EV-harvesting Medium Not specified 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 Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD81 Not detected contaminants Calnexin 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 NTA Report type Size range/distribution Reported size (nm) 1-250

	EV210072	4/5	Homo sapiens	MG63	(d)(U)C	Gong, Liangzhi	2018	33%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 33% (75th 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: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MG63 EV-harvesting Medium Not specified 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 Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Alix/ CD81 Detected contaminants Calnexin 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 NTA Report type Size range/distribution Reported size (nm) 1-200

	EV200147	1/3	Homo sapiens	BeWo	(d)(U)C	Shen, Li	2018	33%
Study summary Full title Placenta‑associated serum exosomal miR‑155 derived from patients with preeclampsia inhibits eNOS expression in human umbilical vein endothelial cells All authors Li Shen, Yujing Li, Ruotian Li, Zhenyu Diao, Muyi Yany, Mengfei Wu, Haixiang Sun, Guijun Yan, Yali Hu Journal Int J Mol Med Abstract Preeclampsia (PE) is considered to be initiated by abnormal placentation in early pregnancy and resu (show more...)Preeclampsia (PE) is considered to be initiated by abnormal placentation in early pregnancy and results in systemic endothelial cell dysfunction in the second or third trimester. MicroRNAs (miRs) expressed in the human placenta can be secreted into maternal circulation via exosomes, which are secreted extracellular vesicles that serve important roles in intercellular communication. The present study hypothesized that upregulation of placenta‑associated serum exosomal miR‑155 from patients with PE may suppress endothelial nitric oxide synthase (eNOS) expression in endothelial cells. The results demonstrated that placenta‑associated serum exosomes from patients with PE decreased nitric oxide (NO) production and eNOS expression in primary human umbilical vein endothelial cells (HUVECs). Subsequently, an upregulation of placenta‑associated serum exosomal miR‑155 was detected in patients with PE compared with in gestational age‑matched normal pregnant women. In addition, the results demonstrated that overexpression of exosomal miR‑155 from BeWo cells was internalized into HUVECs, and was able to suppress eNOS expression by targeting its 3'‑untranslated region. The results of the present study indicated that placenta‑associated serum exosomes may inhibit eNOS expression in endothelial cell during PE development in humans, and this phenomenon may be partly due to increased miR‑155 expression in placenta‑associated serum exosomes. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: "CD81/ PLAP/ CD63" non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells BeWo EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified 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 Not specified Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins "CD63/ PLAP/ CD81" Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV180026	2/7	Homo sapiens	MCF10A	(d)(U)C Filtration	Wenzhe Li	2018	33%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 33% (75th 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 Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF10A EV-harvesting Medium EV-depleted serum 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No

	EV180026	4/7	Homo sapiens	MDAMB468	(d)(U)C Filtration	Wenzhe Li	2018	33%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 33% (75th 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 Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ EpCAM non-EV: Calnexin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB468 EV-harvesting Medium EV-depleted serum 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63, CD81, Flotillin-1, EpCAM Not detected contaminants Calnexin Characterization: Lipid analysis No

	EV180026	6/7	Homo sapiens	Serum	(d)(U)C Filtration	Wenzhe Li	2018	33%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 33% (76th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum 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 Filtration Adj. k-factor 104.6 (pelleting) / 104.6 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ EpCAM non-EV: Albumin Proteomics no Show all info Study aim Function, Biomarker, New methodological development 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: time(min) 1200 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 104.6 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Wash: adjusted k-factor 104.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Albumin Characterization: Lipid analysis No

	EV180030	2/7	Homo sapiens	H9	(d)(U)C qEV	Zhaohao Liao	2018	33%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 33% (75th 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 qEV Protein markers EV: / CD81/ CD63 non-EV: AChE 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 H9 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Other 1 Acetylcholinesterase assay Detected EV-associated proteins Detected contaminants AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV220168	1/2	Homo sapiens	CHLA-258	(d)(U)C	Zhang P	2018	29%
Study summary Full title Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip. All authors Zhang P, Crow J, Lella D, Zhou X, Samuel G, Godwin AK, Zeng Y Journal Lab Chip Abstract Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it rem (show more...)Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring. (hide) EV-METRIC 29% (68th 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: None 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 CHLA-258 EV-harvesting Medium Serum free medium 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: rotor type F40L-8x100 Pelleting: speed (g) 100 000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type F40L-8x100 Wash: speed (g) 110000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127.3 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.08e+11 EM EM-type Scanning-EM Image type Wide-field

	EV220168	2/2	Homo sapiens	CHLA-9	(d)(U)C	Zhang P	2018	29%
Study summary Full title Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip. All authors Zhang P, Crow J, Lella D, Zhou X, Samuel G, Godwin AK, Zeng Y Journal Lab Chip Abstract Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it rem (show more...)Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring. (hide) EV-METRIC 29% (68th 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: None 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 CHLA-9 EV-harvesting Medium Serum free medium 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: rotor type F40L-8x100 Pelleting: speed (g) 100 000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type F40L-8x100 Wash: speed (g) 110000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 152.1 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.17e+12 EM EM-type Scanning-EM Image type Wide-field

	EV220101	1/2	Mus musculus	TSA mammary melanoma	DG (d)(U)C	Diamond JM	2018	29%
Study summary Full title Exosomes Shuttle TREX1-Sensitive IFN-Stimulatory dsDNA from Irradiated Cancer Cells to DCs. All authors Diamond JM, Vanpouille-Box C, Spada S, Rudqvist NP, Chapman JR, Ueberheide BM, Pilones KA, Sarfraz Y, Formenti SC, Demaria S Journal Cancer Immunol Res Abstract Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA ( (show more...)Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. , RT-TEX elicited tumor-specific CD8 T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. . (hide) EV-METRIC 29% (68th 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 Density gradient (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells TSA mammary melanoma EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial 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: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.25 M Highest density fraction 2 M Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 2 Orientation Bottom-up Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No

	EV220101	2/2	Mus musculus	TSA mammary melanoma	DG (d)(U)C	Diamond JM	2018	29%
Study summary Full title Exosomes Shuttle TREX1-Sensitive IFN-Stimulatory dsDNA from Irradiated Cancer Cells to DCs. All authors Diamond JM, Vanpouille-Box C, Spada S, Rudqvist NP, Chapman JR, Ueberheide BM, Pilones KA, Sarfraz Y, Formenti SC, Demaria S Journal Cancer Immunol Res Abstract Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA ( (show more...)Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. , RT-TEX elicited tumor-specific CD8 T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. . (hide) EV-METRIC 29% (68th 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 8 Gy irradiation 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 Density gradient (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells TSA mammary melanoma EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial 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: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.25 M Highest density fraction 2 M Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 2 Orientation Bottom-up Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No

	EV210431	1/2	Homo sapiens	Placental perfusate	(d)(U)C	Tersigni C	2018	29%
Study summary Full title HLA-DR is aberrantly expressed at feto-maternal interface in pre-eclampsia. All authors Tersigni C, Redman CW, Dragovic R, Tannetta D, Scambia G, Di Simone N, Sargent I, Vatish M Journal J Reprod Immunol Abstract In normal pregnancy, villous cytotrophoblast and syncytiotrophoblast do not express HLA Class I and (show more...)In normal pregnancy, villous cytotrophoblast and syncytiotrophoblast do not express HLA Class I and Class II molecules, while invasive extravillous trophoblast only express class I HLA-C and the atypical class Ib antigens, HLA-G, -E and -F. Inadequate maternal tolerance of invasive trophoblast has been proposed as a possible immunologic trigger of poor trophoblast invasion and subsequent occurrence of pre-eclampsia. This study aimed to investigate possible aberrant expression of class II HLA-DR on placentae and syncytiotrophoblast-derived extracellular vesicles (STEVs), obtained by dual placental perfusion, from pre-eclampsia (n = 23) and normal pregnant (n = 14) women. Here we demonstrate that HLA-DR can be detected in syncytiotrophoblast from a significant proportion of pre-eclampsia but not control placentae. HLA-DR was also observed, by flow cytometry, on STEVs and associated with placental alkaline phosphatase to validate their placental origin. HLA-DR positive syncytiotrophoblast was detected in placental biopsies from pre-eclampsia but not normal control cases, using immunohistochemistry. The HLA may be fetal or maternal origin. In the latter case a possible mechanism of acquisition is trogocytosis. (hide) EV-METRIC 29% (66th 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 Placental perfusate 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: Placental Alkaline Phosphatase/ HLA-DR/ HLA-ABC/ HLA-G non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Placental perfusate Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type L8-80 M Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Flow cytometry Type of Flow cytometry SRII Flow Cytometer (BD Biosciences) Calibration bead size 1 Antibody details provided? No Detected EV-associated proteins Placental Alkaline Phosphatase/ HLA-DR Not detected EV-associated proteins HLA-ABC/ HLA-G Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type LSRII Flow Cytometer (BD Biosciences) Hardware adjustment Calibration bead size 1

	EV210431	2/2	Homo sapiens	Placental perfusate	(d)(U)C	Tersigni C	2018	29%
Study summary Full title HLA-DR is aberrantly expressed at feto-maternal interface in pre-eclampsia. All authors Tersigni C, Redman CW, Dragovic R, Tannetta D, Scambia G, Di Simone N, Sargent I, Vatish M Journal J Reprod Immunol Abstract In normal pregnancy, villous cytotrophoblast and syncytiotrophoblast do not express HLA Class I and (show more...)In normal pregnancy, villous cytotrophoblast and syncytiotrophoblast do not express HLA Class I and Class II molecules, while invasive extravillous trophoblast only express class I HLA-C and the atypical class Ib antigens, HLA-G, -E and -F. Inadequate maternal tolerance of invasive trophoblast has been proposed as a possible immunologic trigger of poor trophoblast invasion and subsequent occurrence of pre-eclampsia. This study aimed to investigate possible aberrant expression of class II HLA-DR on placentae and syncytiotrophoblast-derived extracellular vesicles (STEVs), obtained by dual placental perfusion, from pre-eclampsia (n = 23) and normal pregnant (n = 14) women. Here we demonstrate that HLA-DR can be detected in syncytiotrophoblast from a significant proportion of pre-eclampsia but not control placentae. HLA-DR was also observed, by flow cytometry, on STEVs and associated with placental alkaline phosphatase to validate their placental origin. HLA-DR positive syncytiotrophoblast was detected in placental biopsies from pre-eclampsia but not normal control cases, using immunohistochemistry. The HLA may be fetal or maternal origin. In the latter case a possible mechanism of acquisition is trogocytosis. (hide) EV-METRIC 29% (66th 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 Placental perfusate Sample origin Pre-eclampsia 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: Placental Alkaline Phosphatase/ HLA-DR/ HLA-ABC/ HLA-G non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Placental perfusate Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type L8-80 M Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Flow cytometry Type of Flow cytometry SRII Flow Cytometer (BD Biosciences) Calibration bead size 1 Antibody details provided? No Detected EV-associated proteins Placental Alkaline Phosphatase/ HLA-DR Not detected EV-associated proteins HLA-ABC/ HLA-G Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type LSRII Flow Cytometer (BD Biosciences) Hardware adjustment Calibration bead size 1 Report type Not Reported

	EV210415	1/2	Homo sapiens	Blood plasma	IAF (d)(U)C ExoQuick	Winston CN	2018	29%
Study summary Full title Growth Hormone-Releasing Hormone Modulation of Neuronal Exosome Biomarkers in Mild Cognitive Impairment. All authors Winston CN, Goetzl EJ, Baker LD, Vitiello MV, Rissman RA Journal J Alzheimers Dis Abstract Age-related changes in cognition are linked to decreased expression of somatotropins, GHRH and IGF-1 (show more...)Age-related changes in cognition are linked to decreased expression of somatotropins, GHRH and IGF-1. Mild cognitive impairment (MCI) and Alzheimer's disease (AD) are heterogeneous conditions. The loss of GHRH signaling in the brain may be mechanistically involved in AD pathogenesis. The consequent need to identify AD at an early and perhaps more treatable stage has fueled research into blood-based, exosome biomarkers. Plasma exosomes from participants enrolled in a randomized, double-blind, placebo-controlled 20-week trial of GHRH administration, were isolated, precipitated, and enriched by immuno-absorption with anti-L1CAM antibody (neural adhesion protein) from adults with MCI and age-matched, cognitively normal controls (CNC). Extracted protein cargo from neuronally-derived exosomes (NDEs) were assessed by ELISAs for protein levels implicated in AD neuropathology and for synaptic proteins altered by AD. Plasma NDE concentrations of Aβ1-42 were significantly increased while plasma NDE concentrations of NRGN, synaptophysin, synaptotagmin, and synaptopodin were significantly decreased in patients with MCI, independent of GHRH treatment. Plasma NDE concentrations of ptau-S396 and GAP43 were not affected by cognitive status (CNC versus MCI) or by GHRH treatment. Aβ1-42, neurogranin (NRGN), synaptophysin, synaptotagmin, and synaptopodin demonstrated the highest diagnostic accuracy for distinguishing between CNC and MCI patients, while synaptophysin and synaptotagmin demonstrated moderate accuracy in distinguishing between placebo-treated and GHRH-treated, MCI patients. (hide) EV-METRIC 29% (60th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles 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 Immunoaffinity capture (non-commercial) (Differential) (ultra)centrifugation Commercial method Protein markers EV: A1-42/ ptsau-S396/ neurogranin/ synaptotagmin-2/ synaptopodin/ GAP43/ CD81 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) CD171 Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? No Detected EV-associated proteins A1-42/ ptsau-S396/ neurogranin/ synaptotagmin-2/ synaptopodin/ GAP43/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210415	2/2	Homo sapiens	Blood plasma	IAF (d)(U)C ExoQuick	Winston CN	2018	29%
Study summary Full title Growth Hormone-Releasing Hormone Modulation of Neuronal Exosome Biomarkers in Mild Cognitive Impairment. All authors Winston CN, Goetzl EJ, Baker LD, Vitiello MV, Rissman RA Journal J Alzheimers Dis Abstract Age-related changes in cognition are linked to decreased expression of somatotropins, GHRH and IGF-1 (show more...)Age-related changes in cognition are linked to decreased expression of somatotropins, GHRH and IGF-1. Mild cognitive impairment (MCI) and Alzheimer's disease (AD) are heterogeneous conditions. The loss of GHRH signaling in the brain may be mechanistically involved in AD pathogenesis. The consequent need to identify AD at an early and perhaps more treatable stage has fueled research into blood-based, exosome biomarkers. Plasma exosomes from participants enrolled in a randomized, double-blind, placebo-controlled 20-week trial of GHRH administration, were isolated, precipitated, and enriched by immuno-absorption with anti-L1CAM antibody (neural adhesion protein) from adults with MCI and age-matched, cognitively normal controls (CNC). Extracted protein cargo from neuronally-derived exosomes (NDEs) were assessed by ELISAs for protein levels implicated in AD neuropathology and for synaptic proteins altered by AD. Plasma NDE concentrations of Aβ1-42 were significantly increased while plasma NDE concentrations of NRGN, synaptophysin, synaptotagmin, and synaptopodin were significantly decreased in patients with MCI, independent of GHRH treatment. Plasma NDE concentrations of ptau-S396 and GAP43 were not affected by cognitive status (CNC versus MCI) or by GHRH treatment. Aβ1-42, neurogranin (NRGN), synaptophysin, synaptotagmin, and synaptopodin demonstrated the highest diagnostic accuracy for distinguishing between CNC and MCI patients, while synaptophysin and synaptotagmin demonstrated moderate accuracy in distinguishing between placebo-treated and GHRH-treated, MCI patients. (hide) EV-METRIC 29% (60th 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 Mild cognitive impairment 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 Immunoaffinity capture (non-commercial) (Differential) (ultra)centrifugation Commercial method Protein markers EV: A1-42/ ptsau-S396/ neurogranin/ synaptotagmin-2/ synaptopodin/ GAP43/ CD81 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) CD171 Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? No Detected EV-associated proteins A1-42/ ptsau-S396/ neurogranin/ synaptotagmin-2/ synaptopodin/ GAP43/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210353	1/1	Homo sapiens	Placental extract	(d)(U)C SEC (non-commercial) Filtration IAF	Burkova EE	2018	29%
Study summary Full title Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins. All authors Burkova EE, Dmitrenok PS, Bulgakov DV, Vlassov VV, Ryabchikova EI, Nevinsky GA Journal IUBMB Life Abstract Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are i (show more...)Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods-centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density ("nonvesicles" 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed/ however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa. This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144-1155, 2018. (hide) EV-METRIC 29% (50th 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 Placental extract 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 Size-exclusion chromatography (non-commercial) Filtration Immunoaffinity capture (non-commercial) Protein markers EV: CD81 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Placental extract Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type JA-30.50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 8 Wash: time (min) 120 Wash: Rotor Type SW 60 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm0.2µm > x > 0.1µm Size-exclusion chromatography Total column volume (mL) 50 Sample volume/column (mL) 0.5 Immunoaffinity capture Selected surface protein(s) CD81 Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Detected EV-associated proteins CD81 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Immuno-EM EM protein CD81 Image type Close-up Report size (nm) 20 - >100 nm

	EV200135	1/4	Homo sapiens	Blood plasma	(d)(U)C	Kovács, Árpád Ferenc	2018	29%
Study summary Full title The impact of circulating preeclampsia-associated extracellular vesicles on the migratory activity and phenotype of THP-1 monocytic cells All authors Árpád Ferenc Kovács, Orsolya Láng, Lilla Turiák, András Ács, László Kőhidai, Nóra Fekete, Bálint Alasztics, Tamás Mészáros, Edit Irén Buzás, János Rigó Jr., Éva Pállinger Journal Sci Rep Abstract Intercellular communication via extracellular vesicles (EVs) and their target cells, especially immu (show more...)Intercellular communication via extracellular vesicles (EVs) and their target cells, especially immune cells, results in functional and phenotype changes that consequently may play a significant role in various physiological states and the pathogenesis of immune-mediated disorders. Monocytes are the most prominent environment-sensing immune cells in circulation, skilled to shape their microenvironments via cytokine secretion and further differentiation. Both the circulating monocyte subset distribution and the blood plasma EV pattern are characteristic for preeclampsia, a pregnancy induced immune-mediated hypertensive disorder. We hypothesized that preeclampsia-associated EVs (PE-EVs) induced functional and phenotypic alterations of monocytes. First, we proved EV binding and uptake by THP-1 cells. Cellular origin and protein cargo of circulating PE-EVs were characterized by flow cytometry and mass spectrometry. An altered phagocytosis-associated molecular pattern was found on 12.5 K fraction of PE-EVs: an elevated CD47 “don’t eat me” signal (p < 0.01) and decreased exofacial phosphatidylserine “eat-me” signal (p < 0.001) were found along with decreased uptake of these PE-EVs (p < 0.05). The 12.5 K fraction of PE-EVs induced significantly lower chemotaxis (p < 0.01) and cell motility but accelerated cell adhesion of THP-1 cells (p < 0.05). The 12.5 K fraction of PE-EVs induced altered monocyte functions suggest that circulating EVs may have a role in the pathogenesis of preeclampsia. (hide) EV-METRIC 29% (60th 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 Healthy pregnant 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: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 15 Pelleting: rotor type 200.88 fixed angle (Hermle) Pelleting: speed (g) 12500 Wash: time (min) 15 Wash: Rotor Type 200.88 fixed angle (Hermle) Wash: speed (g) 12500 EV-subtype Distinction between multiple subtypes pelleting speed Used subtypes 12.5 K pellet (microvesicle enriched) Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis DLS Particle analysis: flow cytometry Flow cytometer type Apogee A50 Micro (Apogee Flow Systems Ltd) Calibration bead size 160 nm; 200 nm; 240nm; 500 nm Report type Size range / distribution Reported size (nm) >300nm

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