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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200189	1/2	Homo sapiens	Cerebrospinal Fluid	(d)(U)C Sonication Filtration UF	Manek, Rachna	2018	44%
Study summary Full title Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal Fluid Following Traumatic Brain Injury All authors Rachna Manek, Ahmed Moghieb, Zhihui Yang, Dhwani Kumar, Firas Kobessiy, George Anis Sarkis, Vijaya Raghavan, Kevin K W Wang Journal Molecular Neurobiology Abstract Recently, there have been emerging interests in the area of microvesicles and exosome (MV/E) release (show more...)Recently, there have been emerging interests in the area of microvesicles and exosome (MV/E) released from brain cells in relation to neurodegenerative diseases. However, only limited studies focused on MV/E released post-traumatic brain injury (TBI) as they highlight on the mechanistic roles of released proteins. This study sought to examine if CSF samples from severe TBI patients contain MV/E with unique protein contents. First, nanoparticle tracking analysis determined MV/E from TBI have a mode of 74-98 nm in diameter, while control CSF MV/E have a mode of 99-104 nm. Also, there are more MV/E were isolated from TBI CSF (27.8-33.6 × 108/mL) than from control CSF (13.1-18.5 × 108/mL). Transmission electron microscopy (TEM) visualization also confirmed characteristic MV/E morphology. Using targeted immunoblotting approach, we observed the presence of several known TBI biomarkers such as αII-spectrin breakdown products (BDPs), GFAP, and its BDPs and UCH-L1 in higher concentrations in MV/E from TBI CSF than their counterparts from control CSF. Furthermore, we found presynaptic terminal protein synaptophysin and known exosome marker Alix enriched in MV/E from human TBI CSF. In parallel, we conducted nRPLC-tandem mass spectrometry-based proteomic analysis of two control and two TBI CSF samples. Ninety-one proteins were identified with high confidence in MV/E from control CSF, whereas 466 proteins were identified in the counterpart from TBI CSF. MV/E isolated from human CSF contain cytoskeletal proteins, neurite-outgrowth related proteins, and synaptic proteins, extracellular matrix proteins, and complement protein C1q subcomponent subunit B. Taken together, following severe TBI, the injured human brain released increased number of extracellular microvesicles/exosomes (MV/E) into CSF. These TBI MV/E contain several known TBI biomarkers and previously undescribed brain protein markers. It is also possible that such TBI-specific MV/E might contain cell to cell communication factors related to both cell death signaling a well as neurodegeneration pathways. (hide) EV-METRIC 44% (92nd 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 Cerebrospinal Fluid 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 Sonication Filtration Ultrafiltration Protein markers EV: Spectrin/ UCH-L1/ Synaptophysin/ GFAP/ Alix non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cerebrospinal Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) Not spec Membrane type Not specified Other Name other separation method Sonication Characterization: Protein analysis Protein Concentration Method No Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Spectrin/ Alix Not detected EV-associated proteins UCH-L1/ Synaptophysin/ GFAP Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 99-104 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV200189	2/2	Homo sapiens	Cerebrospinal Fluid	(d)(U)C Sonication Filtration UF	Manek, Rachna	2018	44%
Study summary Full title Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal Fluid Following Traumatic Brain Injury All authors Rachna Manek, Ahmed Moghieb, Zhihui Yang, Dhwani Kumar, Firas Kobessiy, George Anis Sarkis, Vijaya Raghavan, Kevin K W Wang Journal Molecular Neurobiology Abstract Recently, there have been emerging interests in the area of microvesicles and exosome (MV/E) release (show more...)Recently, there have been emerging interests in the area of microvesicles and exosome (MV/E) released from brain cells in relation to neurodegenerative diseases. However, only limited studies focused on MV/E released post-traumatic brain injury (TBI) as they highlight on the mechanistic roles of released proteins. This study sought to examine if CSF samples from severe TBI patients contain MV/E with unique protein contents. First, nanoparticle tracking analysis determined MV/E from TBI have a mode of 74-98 nm in diameter, while control CSF MV/E have a mode of 99-104 nm. Also, there are more MV/E were isolated from TBI CSF (27.8-33.6 × 108/mL) than from control CSF (13.1-18.5 × 108/mL). Transmission electron microscopy (TEM) visualization also confirmed characteristic MV/E morphology. Using targeted immunoblotting approach, we observed the presence of several known TBI biomarkers such as αII-spectrin breakdown products (BDPs), GFAP, and its BDPs and UCH-L1 in higher concentrations in MV/E from TBI CSF than their counterparts from control CSF. Furthermore, we found presynaptic terminal protein synaptophysin and known exosome marker Alix enriched in MV/E from human TBI CSF. In parallel, we conducted nRPLC-tandem mass spectrometry-based proteomic analysis of two control and two TBI CSF samples. Ninety-one proteins were identified with high confidence in MV/E from control CSF, whereas 466 proteins were identified in the counterpart from TBI CSF. MV/E isolated from human CSF contain cytoskeletal proteins, neurite-outgrowth related proteins, and synaptic proteins, extracellular matrix proteins, and complement protein C1q subcomponent subunit B. Taken together, following severe TBI, the injured human brain released increased number of extracellular microvesicles/exosomes (MV/E) into CSF. These TBI MV/E contain several known TBI biomarkers and previously undescribed brain protein markers. It is also possible that such TBI-specific MV/E might contain cell to cell communication factors related to both cell death signaling a well as neurodegeneration pathways. (hide) EV-METRIC 44% (92nd 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 Cerebrospinal Fluid Sample origin Traumatic brain injury 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 Sonication Filtration Ultrafiltration Protein markers EV: Spectrin/ UCH-L1/ Synaptophysin/ GFAP/ Alix non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cerebrospinal Fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) Not spec Membrane type Not specified Other Name other separation method Sonication Characterization: Protein analysis Protein Concentration Method No Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Spectrin/ UCH-L1/ Synaptophysin/ GFAP/ Alix Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 74-98 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV200147	2/3	Homo sapiens	Serum	"Precipitation (d)(U)C DG Filtration"	Shen, Li	2018	44%
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 44% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Healthy pregnant 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 "Precipitation (Differential) (ultra)centrifugation Density gradient Filtration" Protein markers EV: "CD81/ PLAP/ CD63" non-EV: None Proteomics no EV density (g/ml) 1.09 Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Not specified Number of initial discontinuous layers Not specified Lowest density fraction Not specified Highest density fraction Not specified Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 300 Fraction volume (mL) Not specified Fraction processing Precipitation of all proteins/vesicles Filtration steps 0.22µm or 0.2µm Other Name other separation method "Precipitation Other Name other separation method Filtration" 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 Mean Reported size (nm) 73.74 EM EM-type Transmission-EM Image type Close-up

	EV180026	7/7	Homo sapiens	MCF7	(d)(U)C Filtration	Wenzhe Li	2018	44%
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 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 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 MCF7 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 Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 141.5 ± 15.04 NTA Report type Size range/distribution Reported size (nm) 146.2 ± 60.2 EM EM-type Transmission-EM Image type Close-up EV concentration Yes

	EV180010	1/2	Homo sapiens	Serum	(d)(U)C qEV	Busatto, Sara	2018	44%
Study summary Full title Uptake Profiles of Human Serum Exosomes by Murine and Human Tumor Cells through Combined Use of Colloidal Nanoplasmonics and Flow Cytofluorimetric Analysis All authors Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, Paolo Bergese Journal Anal Chem Abstract Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capi (show more...)Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth. (hide) EV-METRIC 44% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin 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 qEV Adj. k-factor 89.2 (pelleting) Protein markers EV: ANXA11/ CD63 non-EV: GM130/ APOA1 Proteomics no Show all info Study aim Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Serum 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) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 89.20 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, ANXA11 Not detected contaminants GM130, APOA1 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force-EM Image type Wide-field Report size (nm) 30-150

	EV180083	2/2	Mus musculus	5T33MMvt	DG (d)(U)C ExoQuick	Faict S	2018	43%
Study summary Full title Exosomes play a role in multiple myeloma bone disease and tumor development by targeting osteoclasts and osteoblasts. All authors Faict S, Muller J, De Veirman K, De Bruyne E, Maes K, Vrancken L, Heusschen R, De Raeve H, Schots R, Vanderkerken K, Caers J, Menu E. Journal blood cancer j Abstract Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment w (show more...)Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles takes place. This cross-talk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease. In this paper, we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. We found that 5TGM1 sEVs, or 'exosomes', not only enhanced osteoclast activity, they also blocked osteoblast differentiation and functionality in vitro. Mechanistically, we could demonstrate that transfer of DKK-1 led to a reduction in Runx2, Osterix, and Collagen 1A1 in osteoblasts. In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. Blocking exosome secretion using the sphingomyelinase inhibitor GW4869 not only increased cortical bone volume, but also it sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response when GW4869 and bortezomib were combined. Altogether, our results indicate an important role for exosomes in the BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C ExoQuick Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 5T33MMvt EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 10800 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis None Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No EM EM-type EV concentration

	EV180059	2/6	Gut microbiota	Blood plasma	DG SEC	Tulkens J	2018	42%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 42% (74th 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 LPS-positive bacterial EV Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG SEC Protein markers EV: LPS/ OmpA non-EV: Proteomics no EV density (g/ml) 1.141-1.186 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16,5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No

	EV180059	3/6	Gut microbiota	Blood plasma	SEC	Tulkens J	2018	42%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 42% (74th 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 Inflammatory bowel disease Focus vesicles LPS-positive bacterial EV 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 SEC Protein markers EV: LPS/ OmpA non-EV: Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Immuno-EM EM protein LPS Image type Close-up

	EV180059	5/6	Gut microbiota	Blood plasma	SEC	Tulkens J	2018	42%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 42% (74th 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 Therapy-induced intestinal mucositis Focus vesicles LPS-positive bacterial EV 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 SEC Protein markers EV: LPS/ OmpA non-EV: Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Immuno-EM EM protein E. coli LPS Image type Close-up

	EV180039	1/2	Bos taurus	Non-conditioned medium	DG (d)(U)C	Driedonks, Tom	2018	42%
Study summary Full title Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells All authors Tom A P Driedonks, Maarten K Nijen Twilhaar, Esther N M Nolte-'t Hoen Journal J Extracell Vesicles Abstract Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contamin (show more...)Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contaminating extracellular vesicles (EV) containing RNA. Because of a high degree of sequence similarity among homologous non-coding RNAs of mammalian species, residual FCS-RNA in culture medium may interfere in the analysis of EV-RNA released by cultured cells. Recently, doubts have been raised as to whether commonly used protocols for depletion of FCS-EV efficiently remove FCS-RNA. Moreover, technical details in FCS-EV depletion protocols are known to vary between labs, which may lead to inter-study differences in contaminating FCS-RNA levels. Here, we investigated how technical modifications of EV-depletion protocols affect the efficiency with which bovine RNAs are depleted from FCS, and determined the contribution of contaminating bovine RNA to EV-RNA purified from cell cultures. Our data show differences in depletion efficiency between and within various classes of small non-coding RNA. Importantly, we demonstrate that variations in FCS-EV depletion protocols affect both the quantity and type of residual FCS-RNAs in EV-depleted medium. By using optimised FCS-EV depletion protocols combined with methods for high-grade purification of EV the levels of contaminating bovine RNA in EV populations isolated from cell culture medium can be reduced. With illustrative datasets we also demonstrate that the abundance of a specific RNA in cell culture EV can only be determined if measured relative to background levels of this RNA in medium control samples. These data highlight the need for optimisation and validation of existing and novel FCS-EV depletion methods and urge for accurate descriptions of these methods in publications to increase experimental reproducibility. (hide) EV-METRIC 42% (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 Non-conditioned medium Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 253.9 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Non-conditioned 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) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 0.05 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900 - 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 192000 Pelleting: adjusted k-factor 144.0 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine Calibration bead size 0.1,0.2 EV concentration Yes

	EV180039	2/2	Homo sapiens	HEK293T	DG (d)(U)C	Driedonks, Tom	2018	42%
Study summary Full title Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells All authors Tom A P Driedonks, Maarten K Nijen Twilhaar, Esther N M Nolte-'t Hoen Journal J Extracell Vesicles Abstract Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contamin (show more...)Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contaminating extracellular vesicles (EV) containing RNA. Because of a high degree of sequence similarity among homologous non-coding RNAs of mammalian species, residual FCS-RNA in culture medium may interfere in the analysis of EV-RNA released by cultured cells. Recently, doubts have been raised as to whether commonly used protocols for depletion of FCS-EV efficiently remove FCS-RNA. Moreover, technical details in FCS-EV depletion protocols are known to vary between labs, which may lead to inter-study differences in contaminating FCS-RNA levels. Here, we investigated how technical modifications of EV-depletion protocols affect the efficiency with which bovine RNAs are depleted from FCS, and determined the contribution of contaminating bovine RNA to EV-RNA purified from cell cultures. Our data show differences in depletion efficiency between and within various classes of small non-coding RNA. Importantly, we demonstrate that variations in FCS-EV depletion protocols affect both the quantity and type of residual FCS-RNAs in EV-depleted medium. By using optimised FCS-EV depletion protocols combined with methods for high-grade purification of EV the levels of contaminating bovine RNA in EV populations isolated from cell culture medium can be reduced. With illustrative datasets we also demonstrate that the abundance of a specific RNA in cell culture EV can only be determined if measured relative to background levels of this RNA in medium control samples. These data highlight the need for optimisation and validation of existing and novel FCS-EV depletion methods and urge for accurate descriptions of these methods in publications to increase experimental reproducibility. (hide) EV-METRIC 42% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 253.9 (pelleting) 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 HEK293T EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA 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) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 0.05 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900 - 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 192000 Pelleting: adjusted k-factor 144.0 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine Calibration bead size 0.1,0.2 EV concentration Yes

	EV220202	1/2	Homo sapiens	Urine	(d)(U)C Urine Exosome RNA Isolation Kit (Norgen Biotek)	Zhan Y	2018	38%
Study summary Full title Expression signatures of exosomal long non-coding RNAs in urine serve as novel non-invasive biomarkers for diagnosis and recurrence prediction of bladder cancer. All authors Zhan Y, Du L, Wang L, Jiang X, Zhang S, Li J, Yan K, Duan W, Zhao Y, Wang L, Wang Y, Wang C Journal Mol Cancer Abstract Recently, expression signatures of exosomal long non-coding RNAs (lncRNAs) have been proposed as pot (show more...)Recently, expression signatures of exosomal long non-coding RNAs (lncRNAs) have been proposed as potential non-invasive biomarkers for cancer detection. In this study, we aimed to develop a urinary exosome (UE)-derived lncRNA panel for diagnosis and recurrence prediction of bladder cancer (BC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to screen and evaluate the expressions of eight candidate lncRNAs in a training set (208 urine samples) and a validation set (160 urine samples). A panel consisting of three differently expressed lncRNAs (MALAT1, PCAT-1 and SPRY4-IT1) was established for BC diagnosis in the training set, showing an area under the receiver-operating characteristic (ROC) curve (AUC) of 0.854. Subsequently, the performance of the panel was further verified with an AUC of 0.813 in the validation set, which was significantly higher than that of urine cytology (0.619). In addition, Kaplan-Meier analysis suggested that the up-regulation of PCAT-1 and MALAT1 was associated with poor recurrence-free survival (RFS) of non-muscle-invasive BC (NMIBC) (p < 0.001 and p = 0.002, respectively), and multivariate Cox proportional hazards regression analysis revealed that exosomal PCAT-1 overexpression was an independent prognostic factor for the RFS of NMIBC (p = 0.018). Collectively, our findings indicated that UE-derived lncRNAs possessed considerable clinical value in the diagnosis and prognosis of BC. (hide) EV-METRIC 38% (73rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles 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 Commercial method 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 Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Commercial kit Urine Exosome RNA Isolation Kit (Norgen Biotek) Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD81/ CD63 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) 20 nm to 200 nm EM EM-type Transmission-EM Image type Wide-field Report size (nm) 60 to 150 nm

	EV220155	1/3	Homo sapiens	MDAMB231	Exospin	Campos A	2018	38%
Study summary Full title Caveolin-1-containing extracellular vesicles transport adhesion proteins and promote malignancy in breast cancer cell lines. All authors Campos A, Salomon C, Bustos R, Díaz J, Martínez S, Silva V, Reyes C, Díaz-Valdivia N, Varas-Godoy M, Lobos-González L, Quest AF Journal Nanomedicine (Lond) Abstract Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-relate (show more...)Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1/ possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo. (hide) EV-METRIC 38% (79th 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 Commercial method Protein markers EV: CD9/ Alix/ TSG101/ CAV1/ beta-actin non-EV: Calnexin Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Commercial kit Exospin Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CAV1/ CD9/ Alix/ TSG101 Not detected EV-associated proteins Beta-actin Not detected contaminants Calnexin Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EM Image type Wide-field Report size (nm) 50-100

	EV220155	2/3	Homo sapiens	MDAMB231	Exospin	Campos A	2018	38%
Study summary Full title Caveolin-1-containing extracellular vesicles transport adhesion proteins and promote malignancy in breast cancer cell lines. All authors Campos A, Salomon C, Bustos R, Díaz J, Martínez S, Silva V, Reyes C, Díaz-Valdivia N, Varas-Godoy M, Lobos-González L, Quest AF Journal Nanomedicine (Lond) Abstract Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-relate (show more...)Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1/ possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo. (hide) EV-METRIC 38% (79th 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 shC 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 Commercial method Protein markers EV: CD9/ Alix/ TSG101/ CAV1/ beta-actin non-EV: Calnexin Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Commercial kit Exospin Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CAV1/ CD9/ Alix/ TSG101 Not detected EV-associated proteins Beta-actin Not detected contaminants Calnexin Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EM Image type Wide-field Report size (nm) 50-100

	EV220155	3/3	Homo sapiens	MDAMB231	Exospin	Campos A	2018	38%
Study summary Full title Caveolin-1-containing extracellular vesicles transport adhesion proteins and promote malignancy in breast cancer cell lines. All authors Campos A, Salomon C, Bustos R, Díaz J, Martínez S, Silva V, Reyes C, Díaz-Valdivia N, Varas-Godoy M, Lobos-González L, Quest AF Journal Nanomedicine (Lond) Abstract Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-relate (show more...)Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1/ possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo. (hide) EV-METRIC 38% (79th 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 shCAV1 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 Commercial method Protein markers EV: CD9/ Alix/ TSG101/ CAV1/ beta-actin non-EV: Calnexin Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Commercial kit Exospin Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CD9/ Alix/ TSG101 Not detected EV-associated proteins Beta-actin/ CAV1 Not detected contaminants Calnexin Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EM Image type Wide-field Report size (nm) 50-100

	EV220070	2/3	Homo sapiens	Serum	(d)(U)C qEV	An M	2018	38%
Study summary Full title Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum. All authors An M, Wu J, Zhu J, Lubman DM Journal J Proteome Res Abstract Exosomes are nanosized vesicles that are abundant in biological fluids. In recent years, exosomes ha (show more...)Exosomes are nanosized vesicles that are abundant in biological fluids. In recent years, exosomes have attracted increasing attention as their cargo may provide promising biomarkers for the early diagnosis of and therapy for many diseases, such as cancer. In addition to ultracentrifugation (UC), many alternative methods including size-exclusion chromatography (SEC) have been developed for isolating exosomes. It has been reported that the SEC method provided improved performance relative to the UC method in isolating exosomes from plasma, where the former contained less residual blood protein contamination. We have compared the SEC method with an optimized UC method in isolating exosomes from human serum. This was based on dilution of the serum to reduce the viscosity and a prolonged cycle of UC, followed by another four cycles. We found that >95% of serum proteins were removed without a significant loss of exosome proteins relative to SEC. We also combined one cycle of UC with SEC and found that this method provided improved results relative to the SEC method, although the serum protein contamination was several times higher than that of our optimized UC method. The TEM showed that the size distribution of exosomes isolated from each of the three methods was similar. (hide) EV-METRIC 38% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: ALBU/ CD63 non-EV: Calnexin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ ALBU Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.30e+9 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 77

	EV220058	4/5	Homo sapiens	Blood plasma	ExoQuick	Lee CH	2018	38%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: TSPAN1/ CD63/ CD9/ TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ TSPAN1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220058	5/5	Homo sapiens	Blood plasma	ExoQuick	Lee CH	2018	38%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 38% (72nd 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 Colon 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 Commercial method Protein markers EV: TSPAN1/ CD63/ CD9/ TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ TSPAN1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220057	1/6	Homo sapiens	PCI-13	(d)(U)C SEC (non-commercial)	Ludwig S	2018	38%
Study summary Full title Molecular and Functional Profiles of Exosomes From HPV(+) and HPV(-) Head and Neck Cancer Cell Lines. All authors Ludwig S, Sharma P, Theodoraki MN, Pietrowska M, Yerneni SS, Lang S, Ferrone S, Whiteside TL Journal Front Oncol Abstract Exosomes produced by tumor cells have been shown to reprogram functions of human immune cells. Molec (show more...)Exosomes produced by tumor cells have been shown to reprogram functions of human immune cells. Molecular cargos of exosomes isolated from supernatants of HPV(+) and HPV(-) head and neck cancer (HNC) cell lines or from HNC patients' plasma were compared. The exosome protein profiles resembled those of respective parent tumor cells. Only HPV(+) exosomes carried E6/E7, p16, and survivin. HPV(-) exosomes were negative for cyclin D1 and carried low p53 levels. Immunomodulatory molecules (TGF-β, FasL, OX40, OX40L, and HSP70) were carried by HPV(+) and HPV(-) exosomes. These exosomes co-incubated with human T cells induced apoptosis and suppressed T cell activation and proliferation. HPV(-) exosomes suppressed DC maturation and expression of antigen processing machinery (APM) components. In contrast, HPV(+) exosomes promoted DC maturation and did not suppress expression of APM components in mature DCs. While DCs readily internalized exosomes, T lymphocytes resisted their uptake during the initial 12 h co-culture. Thus, HPV(+) exosomes capable of sustaining DC functions may play a key role in promoting anti-tumor immune responses thereby improving outcome in patients with HPV(+) cancers. (hide) EV-METRIC 38% (79th 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 Size-exclusion chromatography (non-commercial) Protein markers EV: cyclin D1/ p53/ HPV16E6/ HPV16E7/ p16/ / Rb/ OXA40/ OX40L/ LAP-TGFbeta/ FasL/ PTPN11/ survivin/ HSP70/ TSG101 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PCI-13 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 No Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5-1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Rb/ OXA40/ OX40L/ LAP-TGFbeta/ FasL/ PTPN11/ survivin/ HSP70/ TSG101 Not detected EV-associated proteins cyclin D1/ p53/ HPV16E6/ HPV16E7/ p16/ Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 30-150 EM EM-type Transmission-EM Image type Wide-field

	EV220057	3/6	Homo sapiens	UM-SCC-47	(d)(U)C SEC (non-commercial)	Ludwig S	2018	38%
Study summary Full title Molecular and Functional Profiles of Exosomes From HPV(+) and HPV(-) Head and Neck Cancer Cell Lines. All authors Ludwig S, Sharma P, Theodoraki MN, Pietrowska M, Yerneni SS, Lang S, Ferrone S, Whiteside TL Journal Front Oncol Abstract Exosomes produced by tumor cells have been shown to reprogram functions of human immune cells. Molec (show more...)Exosomes produced by tumor cells have been shown to reprogram functions of human immune cells. Molecular cargos of exosomes isolated from supernatants of HPV(+) and HPV(-) head and neck cancer (HNC) cell lines or from HNC patients' plasma were compared. The exosome protein profiles resembled those of respective parent tumor cells. Only HPV(+) exosomes carried E6/E7, p16, and survivin. HPV(-) exosomes were negative for cyclin D1 and carried low p53 levels. Immunomodulatory molecules (TGF-β, FasL, OX40, OX40L, and HSP70) were carried by HPV(+) and HPV(-) exosomes. These exosomes co-incubated with human T cells induced apoptosis and suppressed T cell activation and proliferation. HPV(-) exosomes suppressed DC maturation and expression of antigen processing machinery (APM) components. In contrast, HPV(+) exosomes promoted DC maturation and did not suppress expression of APM components in mature DCs. While DCs readily internalized exosomes, T lymphocytes resisted their uptake during the initial 12 h co-culture. Thus, HPV(+) exosomes capable of sustaining DC functions may play a key role in promoting anti-tumor immune responses thereby improving outcome in patients with HPV(+) cancers. (hide) EV-METRIC 38% (79th 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 Size-exclusion chromatography (non-commercial) Protein markers EV: OXA40/ OX40L/ LAP-TGFbeta/ FasL/ PTPN11/ survivin/ HSP70/ TSG101/ Rb/ cyclin D1/ p53/ HPV16E6/ HPV16E7/ p16 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UM-SCC-47 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 No Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5-1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins OXA40/ OX40L/ LAP-TGFbeta/ FasL/ PTPN11/ survivin/ HSP70/ TSG101/ Rb/ cyclin D1/ p53/ HPV16E6/ HPV16E7/ p16 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 30-150 EM EM-type Transmission-EM Image type Wide-field

	EV210506	3/6	Mus musculus	Mixed cortical neurons/astrocytes	IAF	Ko J	2018	38%
Study summary Full title Diagnosis of traumatic brain injury using miRNA signatures in nanomagnetically isolated brain-derived extracellular vesicles. All authors Ko J, Hemphill M, Yang Z, Sewell E, Na YJ, Sandsmark DK, Haber M, Fisher SA, Torre EA, Svane KC, Omelchenko A, Firestein BL, Diaz-Arrastia R, Kim J, Meaney DF, Issadore D Journal Lab Chip Abstract The accurate diagnosis and clinical management of traumatic brain injury (TBI) is currently limited (show more...)The accurate diagnosis and clinical management of traumatic brain injury (TBI) is currently limited by the lack of accessible molecular biomarkers that reflect the pathophysiology of this heterogeneous disease. To address this challenge, we developed a microchip diagnostic that can characterize TBI more comprehensively using the RNA found in brain-derived extracellular vesicles (EVs). Our approach measures a panel of EV miRNAs, processed with machine learning algorithms to capture the state of the injured and recovering brain. Our diagnostic combines surface marker-specific nanomagnetic isolation of brain-derived EVs, biomarker discovery using RNA sequencing, and machine learning processing of the EV miRNA cargo to minimally invasively measure the state of TBI. We achieved an accuracy of 99% identifying the signature of injured vs. sham control mice using an independent blinded test set (N = 77), where the injured group consists of heterogeneous populations (injury intensity, elapsed time since injury) to model the variability present in clinical samples. Moreover, we successfully predicted the intensity of the injury, the elapsed time since injury, and the presence of a prior injury using independent blinded test sets (N = 82). We demonstrated the translatability in a blinded test set by identifying TBI patients from healthy controls (AUC = 0.9, N = 60). This approach, which can detect signatures of injury that persist across a variety of injury types and individual responses to injury, more accurately reflects the heterogeneity of human TBI injury and recovery than conventional diagnostics, opening new opportunities to improve treatment of traumatic brain injuries. (hide) EV-METRIC 38% (79th 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 Immunoaffinity capture (non-commercial) Protein markers EV: Alix/ TSG101/ CD9/ GluR2 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Mixed cortical neurons/astrocytes Separation Method Immunoaffinity capture Selected surface protein(s) GluR1/GluR2 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101/ GluR2 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus EM EM-type Scanning-EM Image type Close-up Report size (nm) 150-200

	EV210492	3/8	Homo sapiens	Serum	(d)(U)C ExoQuick	Kim DK	2018	38%
Study summary Full title Mastocytosis-derived extracellular vesicles exhibit a mast cell signature, transfer KIT to stellate cells, and promote their activation. All authors Kim DK, Cho YE, Komarow HD, Bandara G, Song BJ, Olivera A, Metcalfe DD Journal Proc Natl Acad Sci U S A Abstract Extracellular vesicles (EVs) have been implicated in the development and progression of hematologica (show more...)Extracellular vesicles (EVs) have been implicated in the development and progression of hematological malignancies. We thus examined serum samples from patients with systemic mastocytosis (SM) and found EVs with a mast cell signature including the presence of tryptase, FcεRI, MRGX2, and KIT. The concentration of these EVs correlated with parameters of disease including levels of serum tryptase, IL-6, and alkaline phosphatase and physical findings including hepatosplenomegaly. Given reports that EVs from one cell type may influence another cell's behavior, we asked whether SM-EVs might affect hepatic stellate cells (HSCs), based on the abnormal liver pathology associated with mastocytosis. We found that KIT was transferred from SM-EVs into an HSC line eliciting proliferation, cytokine production, and differentiation, processes that have been associated with liver pathology. These effects were reduced by KIT inhibition or neutralization and recapitulated by enforced expression of KIT or constitutively active D816V-KIT, a gain-of-function variant associated with SM. Furthermore, HSCs in liver from mice injected with SM-EVs had increased expression of α-SMA and human KIT, particularly around portal areas, compared with mice injected with EVs from normal individuals, suggesting that SM-EVs can also initiate HSC activation in vivo. Our data are thus consistent with the conclusion that SM-EVs have the potential to influence cells outside the hematological compartment and that therapeutic approaches for treatment of SM may be effective in part through inhibition of effects of EVs on target tissues, findings important both to understanding complex disease pathology and in developing interventional agents for the treatment of hematologic diseases. (hide) EV-METRIC 38% (82nd 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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: Heparin/ KIT/ KIT p-Y730/ FcR1 gamma/ FcR1 alpha/ Tryptase/ MRGX2/ Histamine/ Prohibitin/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ KIT/ KIT p-Y730/ FcR1 gamma/ FcR1 alpha/ Tryptase/ MRGX2 Not detected EV-associated proteins Prohibitin ELISA Antibody details provided? No Detected EV-associated proteins Histamine/ Heparin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 93 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210492	5/8	Homo sapiens	Serum	(d)(U)C ExoQuick	Kim DK	2018	38%
Study summary Full title Mastocytosis-derived extracellular vesicles exhibit a mast cell signature, transfer KIT to stellate cells, and promote their activation. All authors Kim DK, Cho YE, Komarow HD, Bandara G, Song BJ, Olivera A, Metcalfe DD Journal Proc Natl Acad Sci U S A Abstract Extracellular vesicles (EVs) have been implicated in the development and progression of hematologica (show more...)Extracellular vesicles (EVs) have been implicated in the development and progression of hematological malignancies. We thus examined serum samples from patients with systemic mastocytosis (SM) and found EVs with a mast cell signature including the presence of tryptase, FcεRI, MRGX2, and KIT. The concentration of these EVs correlated with parameters of disease including levels of serum tryptase, IL-6, and alkaline phosphatase and physical findings including hepatosplenomegaly. Given reports that EVs from one cell type may influence another cell's behavior, we asked whether SM-EVs might affect hepatic stellate cells (HSCs), based on the abnormal liver pathology associated with mastocytosis. We found that KIT was transferred from SM-EVs into an HSC line eliciting proliferation, cytokine production, and differentiation, processes that have been associated with liver pathology. These effects were reduced by KIT inhibition or neutralization and recapitulated by enforced expression of KIT or constitutively active D816V-KIT, a gain-of-function variant associated with SM. Furthermore, HSCs in liver from mice injected with SM-EVs had increased expression of α-SMA and human KIT, particularly around portal areas, compared with mice injected with EVs from normal individuals, suggesting that SM-EVs can also initiate HSC activation in vivo. Our data are thus consistent with the conclusion that SM-EVs have the potential to influence cells outside the hematological compartment and that therapeutic approaches for treatment of SM may be effective in part through inhibition of effects of EVs on target tissues, findings important both to understanding complex disease pathology and in developing interventional agents for the treatment of hematologic diseases. (hide) EV-METRIC 38% (82nd 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 Systemic mastocytosis 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 Commercial method Protein markers EV: CD63/ Histamine/ Prohibitin/ Heparine/ KIT/ KIT p-Y730/ FcR1 gamma/ FcR1 alpha/ Tryptase/ MRGX2/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins KIT/ KIT p-Y730/ FcR1 gamma/ FcR1 alpha/ Tryptase/ MRGX2/ CD9/ CD63 Not detected EV-associated proteins Prohibitin ELISA Antibody details provided? No Detected EV-associated proteins Histamine/ Heparine Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95/ 97 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210442	1/3	Homo sapiens	Caco2	ExoQuick Filtration	Yang YN	2018	38%
Study summary Full title Predictive role of UCA1-containing exosomes in cetuximab-resistant colorectal cancer. All authors Yang YN, Zhang R, Du JW, Yuan HH, Li YJ, Wei XL, Du XX, Jiang SL, Han Y Journal Cancer Cell Int Abstract Primary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic color (show more...)Primary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic colorectal cancer (mCRC) patients. In many cancers, the key role of the long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) in anticancer drug resistance has been confirmed. Emerging evidence has shown that specific exosomal lncRNAs may serve as meaningful biomarkers. In this study, we hypothesize that exosomal UCA1 might predict the response to cetuximab in CRC patients. (hide) EV-METRIC 38% (79th 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 Commercial method Filtration Protein markers EV: TSG101/ Alix/ CD81 non-EV: Tubulin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Caco2 Separation Method Filtration steps 0.45µm > x > 0.22µm, Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Other/ Spectrophotometry Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins TSG101/ Alix/ CD81 Not detected contaminants Tubulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV210442	2/3	Homo sapiens	Caco2	ExoQuick Filtration	Yang YN	2018	38%
Study summary Full title Predictive role of UCA1-containing exosomes in cetuximab-resistant colorectal cancer. All authors Yang YN, Zhang R, Du JW, Yuan HH, Li YJ, Wei XL, Du XX, Jiang SL, Han Y Journal Cancer Cell Int Abstract Primary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic color (show more...)Primary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic colorectal cancer (mCRC) patients. In many cancers, the key role of the long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) in anticancer drug resistance has been confirmed. Emerging evidence has shown that specific exosomal lncRNAs may serve as meaningful biomarkers. In this study, we hypothesize that exosomal UCA1 might predict the response to cetuximab in CRC patients. (hide) EV-METRIC 38% (79th 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 Cetuximab-resistant clone 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 Commercial method Filtration Protein markers EV: TSG101/ Alix/ CD81 non-EV: Tubulin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Caco2 Separation Method Filtration steps 0.45µm > x > 0.22µm, Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Other/ Spectrophotometry Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins TSG101/ Alix/ CD81 Not detected contaminants Tubulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV210421	1/2	Mus musculus	Blood plasma	(d)(U)C ExoQuick	Cho YE	2018	38%
Study summary Full title Exogenous exosomes from mice with acetaminophen-induced liver injury promote toxicity in the recipient hepatocytes and mice. All authors Cho YE, Seo W, Kim DK, Moon PG, Kim SH, Lee BH, Song BJ, Baek MC Journal Sci Rep Abstract Exosomes are small extracellular membrane vesicles released from endosomes of various cells and coul (show more...)Exosomes are small extracellular membrane vesicles released from endosomes of various cells and could be found in most body fluids. The main functions of exosomes have been recognized as important mediators of intercellular communication and as potential biomarkers of various disease states. This study investigated whether exogenous exosomes from mice with acetaminophen (APAP)-induced liver injury can damage the recipient hepatic cells or promote hepatotoxicity in mice. We observed that exogenous exosomes derived from APAP-exposed mice were internalized into the primary mouse hepatocytes or HepG2 hepatoma cells and significantly decreased the viability of these recipient cells. They also elevated mRNA transcripts and proteins associated with the cell death signaling pathways in primary hepatocytes or HepG2 cells via exosomes-to-cell communications. In addition, confocal microscopy of ex vivo liver section showed that exogenously added exosomes were accumulated in recipient hepatocytes. Furthermore, plasma reactive oxygen species and hepatic TNF-α/IL-1β production were elevated in APAP-exosomes recipient mice compared to control-exosomes recipient mice. The levels of apoptosis-related proteins such as phospho-JNK/JNK, Bax, and cleaved caspase-3 were increased in mouse liver received APAP-exosomes. These results demonstrate that exogenous exosomes from APAP-exposed mice with acute liver injury are functional and stimulate cell death or toxicity of the recipient hepatocytes and mice. (hide) EV-METRIC 38% (72nd 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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: TSG101/ CD63/ Arg-1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus 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 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101 Not detected EV-associated proteins Arg-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 87 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210421	2/2	Mus musculus	Blood plasma	(d)(U)C ExoQuick	Cho YE	2018	38%
Study summary Full title Exogenous exosomes from mice with acetaminophen-induced liver injury promote toxicity in the recipient hepatocytes and mice. All authors Cho YE, Seo W, Kim DK, Moon PG, Kim SH, Lee BH, Song BJ, Baek MC Journal Sci Rep Abstract Exosomes are small extracellular membrane vesicles released from endosomes of various cells and coul (show more...)Exosomes are small extracellular membrane vesicles released from endosomes of various cells and could be found in most body fluids. The main functions of exosomes have been recognized as important mediators of intercellular communication and as potential biomarkers of various disease states. This study investigated whether exogenous exosomes from mice with acetaminophen (APAP)-induced liver injury can damage the recipient hepatic cells or promote hepatotoxicity in mice. We observed that exogenous exosomes derived from APAP-exposed mice were internalized into the primary mouse hepatocytes or HepG2 hepatoma cells and significantly decreased the viability of these recipient cells. They also elevated mRNA transcripts and proteins associated with the cell death signaling pathways in primary hepatocytes or HepG2 cells via exosomes-to-cell communications. In addition, confocal microscopy of ex vivo liver section showed that exogenously added exosomes were accumulated in recipient hepatocytes. Furthermore, plasma reactive oxygen species and hepatic TNF-α/IL-1β production were elevated in APAP-exosomes recipient mice compared to control-exosomes recipient mice. The levels of apoptosis-related proteins such as phospho-JNK/JNK, Bax, and cleaved caspase-3 were increased in mouse liver received APAP-exosomes. These results demonstrate that exogenous exosomes from APAP-exposed mice with acute liver injury are functional and stimulate cell death or toxicity of the recipient hepatocytes and mice. (hide) EV-METRIC 38% (72nd 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 Acetaminophen 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 Commercial method Protein markers EV: TSG101/ CD63/ Arg-1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus 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 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ Arg-1/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 92 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210277	3/4	Homo sapiens	Urine	Aqueous two-phase system	Shin H	2018	38%
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 38% (73rd 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 Aqueous two-phase system 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 Other Name other separation method Aqueous two-phase system Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes 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

	EV200128	1/2	Homo sapiens	Serum	(d)(U)C ExoQuick	Jia, Linyan	2018	38%
Study summary Full title Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration All authors Linyan Jia, Xinyao Zhou, Xiaojie Huang, Xianghong Xu, Yuanhui Jia, Yingting Wu, Julei Yao, Yanming Wu, Kai Wang Journal FASEB J Abstract We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulatio (show more...)We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulation of angiogenesis. We report here that both maternal exosomes (MEs) and umbilical exosomes (UEs) significantly enhance HUVEC proliferation, migration, and tube formation. Importantly, ME-treated HUVECs (MEXs) displayed significantly increased migration, but not proliferation or tube formation, compared with UE-treated HUVECs (UEXs). We found that the expression of a subset of migration-related microRNAs (miRNAs), including miR-210-3p, miR-376c-3p, miR-151a-5p, miR-296-5p, miR-122-5p, and miR-550a-5p, among others, were significantly increased or decreased in UEs, and this altered expression was likely correlated with the differential regulation of HUVEC migration. We also found that the mRNA expression of hepatocyte growth factor (HGF) was up-regulated in MEXs and UEXs and, moreover, that inhibiting HGF partially abolished the enhanced cell migration induced by UEs. Our results suggest that both MEs and UEs greatly enhanced endothelial cell (EC) functions and differentially regulated EC migration, which was mostly attributed to the different expression profiles of exosomal miRNA. These findings highlight the importance of exosomes in the regulation of angiogenesis during pregnancy. Exosomal miRNAs, in particular, may be of great significance for the regulation of angiogenesis in maintaining normal pregnancy. (hide) EV-METRIC 38% (82nd 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 Healthy pregnant Focus vesicles Exosomes 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 Commercial method Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ HSP70/ 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 NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-150

	EV200128	2/2	Homo sapiens	Serum	(d)(U)C ExoQuick	Jia, Linyan	2018	38%
Study summary Full title Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration All authors Linyan Jia, Xinyao Zhou, Xiaojie Huang, Xianghong Xu, Yuanhui Jia, Yingting Wu, Julei Yao, Yanming Wu, Kai Wang Journal FASEB J Abstract We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulatio (show more...)We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulation of angiogenesis. We report here that both maternal exosomes (MEs) and umbilical exosomes (UEs) significantly enhance HUVEC proliferation, migration, and tube formation. Importantly, ME-treated HUVECs (MEXs) displayed significantly increased migration, but not proliferation or tube formation, compared with UE-treated HUVECs (UEXs). We found that the expression of a subset of migration-related microRNAs (miRNAs), including miR-210-3p, miR-376c-3p, miR-151a-5p, miR-296-5p, miR-122-5p, and miR-550a-5p, among others, were significantly increased or decreased in UEs, and this altered expression was likely correlated with the differential regulation of HUVEC migration. We also found that the mRNA expression of hepatocyte growth factor (HGF) was up-regulated in MEXs and UEXs and, moreover, that inhibiting HGF partially abolished the enhanced cell migration induced by UEs. Our results suggest that both MEs and UEs greatly enhanced endothelial cell (EC) functions and differentially regulated EC migration, which was mostly attributed to the different expression profiles of exosomal miRNA. These findings highlight the importance of exosomes in the regulation of angiogenesis during pregnancy. Exosomal miRNAs, in particular, may be of great significance for the regulation of angiogenesis in maintaining normal pregnancy. (hide) EV-METRIC 38% (82nd 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 Healthy pregnant; Umbilical cord blood Focus vesicles Exosomes 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 Commercial method Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ HSP70/ 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 NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-150

	EV210366	2/6	Homo sapiens	Primary Lymphatic Endothelial Cells	(d)(U)C ExoQuick Filtration DG	Brown M	2018	37%
Study summary Full title Lymphatic exosomes promote dendritic cell migration along guidance cues. All authors Brown M, Johnson LA, Leone DA, Majek P, Vaahtomeri K, Senfter D, Bukosza N, Schachner H, Asfour G, Langer B, Hauschild R, Parapatics K, Hong YK, Bennett KL, Kain R, Detmar M, Sixt M, Jackson DG, Kerjaschki D Journal J Cell Biol Abstract Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendri (show more...)Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments. (hide) EV-METRIC 37% (79th 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-rich endothelial vesicles 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 Commercial method Filtration Density gradient Protein markers EV: CD63 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary Lymphatic Endothelial Cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type P40ST (Hitachi) Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210366	4/6	Homo sapiens	Primary Lymphatic Endothelial Cells	(d)(U)C ExoQuick Filtration DG	Brown M	2018	37%
Study summary Full title Lymphatic exosomes promote dendritic cell migration along guidance cues. All authors Brown M, Johnson LA, Leone DA, Majek P, Vaahtomeri K, Senfter D, Bukosza N, Schachner H, Asfour G, Langer B, Hauschild R, Parapatics K, Hong YK, Bennett KL, Kain R, Detmar M, Sixt M, Jackson DG, Kerjaschki D Journal J Cell Biol Abstract Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendri (show more...)Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments. (hide) EV-METRIC 37% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin TNF- containing EV harvesting media (7ng/mL) Focus vesicles Exosome-rich endothelial vesicles 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 Commercial method Filtration Density gradient Protein markers EV: CD63 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary Lymphatic Endothelial Cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type P40ST (Hitachi) Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210266	1/8	Homo sapiens	MDA-MB-231	(d)(U)C ExoQuick	Lee TS	2018	37%
Study summary Full title Facile metabolic glycan labeling strategy for exosome tracking. All authors Lee TS, Kim Y, Zhang W, Song IH, Tung CH Journal Biochim Biophys Acta Gen Subj Abstract Exosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surroundi (show more...)Exosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surrounding plasma membrane. Exosomes have various diagnostic and therapeutic potentials in cancer and other diseases, thus tracking exosomes is an important issue. (hide) EV-METRIC 37% (79th 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 Commercial method Protein markers EV: CD63/ ITGA6/ Beta-actin non-EV: None Proteomics no Show all info Study aim New methodological development/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ ITGA6/ Beta-actin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean EM EM-type Transmission-EM Image type Close-up Report type Not Reported EV-concentration No

	EV210266	5/8	Homo sapiens	MCF7	(d)(U)C ExoQuick	Lee TS	2018	37%
Study summary Full title Facile metabolic glycan labeling strategy for exosome tracking. All authors Lee TS, Kim Y, Zhang W, Song IH, Tung CH Journal Biochim Biophys Acta Gen Subj Abstract Exosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surroundi (show more...)Exosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surrounding plasma membrane. Exosomes have various diagnostic and therapeutic potentials in cancer and other diseases, thus tracking exosomes is an important issue. (hide) EV-METRIC 37% (79th 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 Commercial method Protein markers EV: CD63/ ITGA6/ Beta-actin non-EV: None Proteomics no Show all info Study aim New methodological development/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ ITGA6/ Beta-actin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean EM EM-type Transmission-EM Image type Close-up Report type Not Reported EV-concentration No

	EV180004	1/1	Homo sapiens	Blood plasma	(d)(U)C qEV	Picciolini S	2018	37%
Study summary Full title Detection and Characterization of Different Brain-Derived Subpopulations of Plasma Exosomes by Surface Plasmon Resonance Imaging. All authors Picciolini S, Gualerzi A, Vanna R, Sguassero A, Gramatica F, Bedoni M, Masserini M, Morasso C Journal Anal Chem Abstract The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealin (show more...)The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases. (hide) EV-METRIC 37% (70th 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 (d)(U)C qEV Protein markers EV: CD81/ ephrinB/ CD171/ PLP1/ Flotillin-1/ CD9 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 10,000 g and 50,000 g Pelleting performed No Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 59.77 Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD81, Flotillin-1 Other 1 Surface plasmon resonance imaging Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield 1500000000 EM EM-type Transmission-EM Image type Wide-field

	EV170014	3/4	Homo sapiens	HEK293	(d)(U)C Filtration SEC	Dionysios C Watson	2018	37%
Study summary Full title Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes All authors Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis Journal J Extracell Vesicles Abstract The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide) EV-METRIC 37% (79th 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 human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected 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 SEC Protein markers EV: hetIL-15/Lactadherin non-EV: ferritin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Size-exclusion chromatography Total column volume (mL) 120 Sample volume/column (mL) 5 Resin type Superdex 200 Characterization: Protein analysis Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes Particle yield 240000000000

	EV210027	1/2	Homo sapiens	Primary adipose-derived stem cells	(d)(U)C Filtration	Han, Yu-di	2018	34%
Study summary Full title Co-transplantation of exosomes derived from hypoxia-preconditioned adipose mesenchymal stem cells promotes neovascularization and graft survival in fat grafting All authors Yu-di Han, Yun Bai, Xin-Long Yan, Jing Ren, Quan Zeng, Xiao-Dong Li, Xue-Tao Pei, Yan Han Journal Biochem Biophys Res Commun Abstract Background: Adipose-derived stromal cells (ADSCs)-derived exosomes (ADSC-Exos) account for the proan (show more...)Background: Adipose-derived stromal cells (ADSCs)-derived exosomes (ADSC-Exos) account for the proangiogenic potential of stem cell. This study aimed to investigate the effect of ADSC-derived exosomes (ADSC-Exos) on the survival in fat grafting. Methods: A nude mouse model of subcutaneous fat grafting was adopted. Hypoxic preconditioned ADSC-Exos and ADSC-Exos were injected around the grafted tissue. The fat graft sample was weighed and examined by hematoxylin and eosin (H&E) staining and immunohistochemistry. Laser Doppler flowmetry and CD31 immunofluorescence staining were used to analyze neovascularization. Results: ADSC-Exo and hypoxic ADSC-Exo groups had a significantly higher weight of fat graft and more perilipin-positive adipocytes than the control groups from 2 to 8 weeks after grafting, and the hypoxic ADSC-Exo group had better outcomes (all P < 0.05). H&E staining showed that ADSC-Exos attenuated infiltration of inflammatory cells around the fat grafts. Laser Doppler flowmetry showed that the two ADSC-Exo groups had better blood perfusion in the graft tissue than the control groups (all P < 0.05). Immunofluorescence demonstrated that the hypoxic ADSC-Exo group had significantly more CD31-positive cells than the ADSC-Exo group. In vitro study showed that hypoxic ADSC-Exos treatment significantly increased the migration (at 12 and 24 h) and in vitro capillary network formation (at 12 h) in the human umbilical vein endothelial cells (HUVECs) as compared with the ADSC-Exo group and control group (all P < 0.05). Conclusions: Co-transplantation of ADSC-Exos can effectively promote the survival of graft, neovascularization and attenuated inflammation in the fat grafts. Hypoxia treatment can further enhance the beneficial effect of ADSC-Exos. (hide) EV-METRIC 34% (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 Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary adipose-derived stem cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) not specified Wash: time (min) 60 Wash: Rotor Type Type 45 Ti Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 98 EV concentration Yes Particle yield No NA EM EM-type Transmission electron microscopy Image type Wide-field

	EV200136	1/7	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Miranda, Jezid	2018	34%
Study summary Full title Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth All authors Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon Journal Placenta Abstract Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (hide) EV-METRIC 34% (69th 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 Not pregnant 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 Filtration Protein markers EV: PLAP non-EV: None Proteomics no EV density (g/ml) 1.12-1.188g/ml Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Bottom-up Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: volume per fraction 0.05 Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? No Detected EV-associated proteins PLAP Characterization: Lipid analysis No

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