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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180074	1/4	Homo sapiens	MDAMB231	(d)(U)C	You, Sixian	2019	43%
Study summary Full title Label-free visualization and characterization of extracellular vesicles in breast cancer All authors Sixian You, Ronit Barkalifa, Eric J Chaney, Haohua Tu, Jaena Park, Janet Elise Sorrells, Yi Sun, Yuan-Zhi Liu, Lin Yang, Danny Z Chen, Marina Marjanovic, Saurabh Sinha, Stephen A Boppart Journal Proc Natl Acad Sci U S A Abstract Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One (show more...)Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression. (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 (d)(U)C Protein markers EV: non-EV: Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Surespin 630 (36 ml) Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 30 Wash: time (min) 60 Wash: Rotor Type Surespin 630 (36 ml) Wash: speed (g) 10000 EV-subtype Distinction between multiple subtypes Size Used subtypes Medium size EVs 200 nm-2000 nm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 227.4 +/- 1.2 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180074	2/4	Homo sapiens	MDAMB231	(d)(U)C	You, Sixian	2019	43%
Study summary Full title Label-free visualization and characterization of extracellular vesicles in breast cancer All authors Sixian You, Ronit Barkalifa, Eric J Chaney, Haohua Tu, Jaena Park, Janet Elise Sorrells, Yi Sun, Yuan-Zhi Liu, Lin Yang, Danny Z Chen, Marina Marjanovic, Saurabh Sinha, Stephen A Boppart Journal Proc Natl Acad Sci U S A Abstract Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One (show more...)Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression. (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 (d)(U)C Protein markers EV: non-EV: Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Surespin 630 (36 ml) Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 30 Wash: time (min) 60 Wash: Rotor Type Surespin630 (36 ml) Wash: speed (g) 10000 EV-subtype Distinction between multiple subtypes Size Used subtypes Small size EVs < 200 nm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 121.0 +/- 1.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180074	3/4	Homo sapiens	MCF7	(d)(U)C	You, Sixian	2019	43%
Study summary Full title Label-free visualization and characterization of extracellular vesicles in breast cancer All authors Sixian You, Ronit Barkalifa, Eric J Chaney, Haohua Tu, Jaena Park, Janet Elise Sorrells, Yi Sun, Yuan-Zhi Liu, Lin Yang, Danny Z Chen, Marina Marjanovic, Saurabh Sinha, Stephen A Boppart Journal Proc Natl Acad Sci U S A Abstract Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One (show more...)Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression. (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 (d)(U)C Protein markers EV: non-EV: Proteomics no Show all info Study aim New methodological development 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 Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Surespin 630 (36 ml) Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 30 Wash: time (min) 60 Wash: Rotor Type Surespin 630 (36 ml) Wash: speed (g) 10000 EV-subtype Distinction between multiple subtypes Size Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 234.7 +/- 10.7 nm EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180074	4/4	Homo sapiens	MCF10A	(d)(U)C	You, Sixian	2019	43%
Study summary Full title Label-free visualization and characterization of extracellular vesicles in breast cancer All authors Sixian You, Ronit Barkalifa, Eric J Chaney, Haohua Tu, Jaena Park, Janet Elise Sorrells, Yi Sun, Yuan-Zhi Liu, Lin Yang, Danny Z Chen, Marina Marjanovic, Saurabh Sinha, Stephen A Boppart Journal Proc Natl Acad Sci U S A Abstract Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One (show more...)Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression. (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 (d)(U)C Protein markers EV: non-EV: Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF10A EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Surespin 630 (36 ml) Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 30 Wash: time (min) 60 Wash: Rotor Type Surespin 630 (36 ml) Wash: speed (g) 10000 EV-subtype Distinction between multiple subtypes Size Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 324.2 +/- 1.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170033	2/2	Homo sapiens	Blood plasma	SEC	Roura S	2019	42%
Study summary Full title Proteomic signature of circulating extracellular vesicles in dilated cardiomyopathy. All authors Roura S, Gámez-Valero A, Lupón J, Gálvez-Montón C, Borràs FE, Bayes-Genis A. Journal Lab Invest. Abstract Dilated cardiomyopathy (DCM) remains a major cause of heart failure and carries a poor prognosis des (show more...)Dilated cardiomyopathy (DCM) remains a major cause of heart failure and carries a poor prognosis despite important advances in recent years. Better disease characterization using novel molecular techniques is needed to refine its progression. This study explored the proteomic signature of plasma-derived extracellular vesicles (EVs) obtained from DCM patients and healthy controls using size-exclusion chromatography (SEC). EV-enriched fractions were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Raw data obtained from LC-MS/MS were analyzed against the Uniprot human database using MaxQuant software. Additional analyses using Perseus software were based on the Intensity-Based Absolute Quantification (iBAQ) values from MaxQuant analyses. A total of 90.07 ± 21 proteins (227 different proteins) in the DCM group and 96.52 ± 17.91 proteins (183 different proteins) in the control group were identified. A total of 176 proteins (74.6%) were shared by controls and DCM patients, whereas 51 proteins were exclusive for the DCM group and 7 proteins were exclusive for the control group. Fibrinogen (α, β and γ chain), serotransferrin, α-1-antitrypsin, and a variety of apolipoprotein family members (C-I, C-III, D, H or β-2-glycoprotein, and J or clusterin) were clustered in SEC-EVs derived from DCM patients relative to controls (p < 0.05). Regarding Gene Ontology analysis, response to stress and protein activation-related proteins were enriched in DCM-EVs compared with controls. Thus, the present study reports the distinct proteomic signature of circulating DCM-EVs compared with control-EVs. Furthermore, we confirm that SEC obtains highly purified EV fractions from peripheral blood samples for subsequent use in determining disease-specific proteomic signatures. (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 dilated cardiomyopathy 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 SEC Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 15 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis PMID previous EV protein analysis Flow cytometry (after non-specific association of Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-200 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV220056	1/8	Homo sapiens	T98G	(d)(U)C Total Exosome Isolation	Dai X	2019	38%
Study summary Full title AHIF promotes glioblastoma progression and radioresistance via exosomes. All authors Dai X, Liao K, Zhuang Z, Chen B, Zhou Z, Zhou S, Lin G, Zhang F, Lin Y, Miao Y, Li Z, Huang R, Qiu Y, Lin R Journal Int J Oncol Abstract Glioblastoma multiforme (GBM) has the highest mortality rate among patients with brain tumors, and r (show more...)Glioblastoma multiforme (GBM) has the highest mortality rate among patients with brain tumors, and radiotherapy forms an important part of its treatment. Thus, there is an urgent requirement to elucidate the mechanisms conferring GBM progression and radioresistance. In the present study, it was identified that antisense transcript of hypoxia‑inducible factor‑1α (AHIF) was significantly upregulated in GBM cancerous tissues, as well as in radioresistant GBM cells. The expression of AHIF was also upregulated in response to radiation. Knockdown of AHIF in GBM cells decreased viability and invasive capacities, and increased the proportion of apoptotic cells. By contrast, overexpression of AHIF in GBM cells increased viability and invasive capacities, and decreased the proportion of apoptotic cells. Furthermore, exosomes derived from AHIF‑knockdown GBM cells inhibited viability, invasion and radioresistance, whereas exosomes derived from AHIF‑overexpressing GBM cells promoted viability, invasion and radioresistance. Further biochemical analysis identified that AHIF regulates factors associated with migration and angiogenesis in exosomes. To the best of our knowledge, the present study is the first to establish that AHIF promotes glioblastoma progression and radioresistance via exosomes, which suggests that AHIF is a potential therapeutic target for GBM. (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 Commercial method Protein markers EV: CD81/ CD63 non-EV: COX IV Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells T98G Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation 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/ CD81 Not detected contaminants COX IV 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

	EV220056	2/8	Homo sapiens	T98G	(d)(U)C Total Exosome Isolation	Dai X	2019	38%
Study summary Full title AHIF promotes glioblastoma progression and radioresistance via exosomes. All authors Dai X, Liao K, Zhuang Z, Chen B, Zhou Z, Zhou S, Lin G, Zhang F, Lin Y, Miao Y, Li Z, Huang R, Qiu Y, Lin R Journal Int J Oncol Abstract Glioblastoma multiforme (GBM) has the highest mortality rate among patients with brain tumors, and r (show more...)Glioblastoma multiforme (GBM) has the highest mortality rate among patients with brain tumors, and radiotherapy forms an important part of its treatment. Thus, there is an urgent requirement to elucidate the mechanisms conferring GBM progression and radioresistance. In the present study, it was identified that antisense transcript of hypoxia‑inducible factor‑1α (AHIF) was significantly upregulated in GBM cancerous tissues, as well as in radioresistant GBM cells. The expression of AHIF was also upregulated in response to radiation. Knockdown of AHIF in GBM cells decreased viability and invasive capacities, and increased the proportion of apoptotic cells. By contrast, overexpression of AHIF in GBM cells increased viability and invasive capacities, and decreased the proportion of apoptotic cells. Furthermore, exosomes derived from AHIF‑knockdown GBM cells inhibited viability, invasion and radioresistance, whereas exosomes derived from AHIF‑overexpressing GBM cells promoted viability, invasion and radioresistance. Further biochemical analysis identified that AHIF regulates factors associated with migration and angiogenesis in exosomes. To the best of our knowledge, the present study is the first to establish that AHIF promotes glioblastoma progression and radioresistance via exosomes, which suggests that AHIF is a potential therapeutic target for GBM. (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 AHIF knockdown Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD81/ CD63 non-EV: COX IV Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells T98G Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation 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/ CD81 Not detected contaminants COX IV Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV210466	1/3	Homo sapiens	Embryonic lung fibroblast hTERT transformed cells	DG (d)(U)C Dialysis UF after density gradient UF Filtration	Dogrammatzis C	2019	38%
Study summary Full title Biogenesis of Extracellular Vesicles during Herpes Simplex Virus 1 Infection: Role of the CD63 Tetraspanin. All authors Dogrammatzis C, Deschamps T, Kalamvoki M Journal J Virol Abstract Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The vir (show more...)Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The virus primarily infects mucoepithelial cells and establishes latent reservoirs in neurons in sensory ganglia. Frequent reactivation has been linked to severe diseases, especially in immunocompromised individuals. Earlier, we reported that viral and host factors are packaged in extracellular vesicles (EVs) and delivered to uninfected cells, where they activate antiviral responses and restrict virus infection. Here, we interrogated the effect of HSV-1 infection on EV biogenesis. We found that HSV-1 infection causes a decrease in the amount of intracellular CD63 protein with a concomitant increase in extracellular CD63. This observation correlates with our previous finding that infected cells release more CD63-positive EVs than uninfected cells. The stimulation of CD63 exocytosis requires virus replication. CD63 is a member of the tetraspanin family of proteins that traffics between the plasma membrane and endosomal compartments and has a role in sorting cargo into the EVs. Previously, we reported that in cells depleted of CD63, HSV-1 virus yields increased, and here we provide data showing that in cells overexpressing CD63, HSV-1 virus yields decreased. Taken together, our data indicate that CD63 negatively impacts HSV-1 infection and that the CD63-positive EVs could control the dissemination of the virus in the host. Perhaps EV release by HSV-1-infected cells is a mechanism that controls virus dissemination. Intercellular communication, especially in neurons, largely relies on EVs, and modulation of EVs is known to impact physiological processes. Here, we present evidence that HSV-1 infection causes major alterations in the biogenesis of EVs, including an increase in their number and an increase in the CD63-positive population of EVs. These alterations result in an enrichment of the milieu of infection with EVs carrying signatures from infected cells. In addition to changes in the origin and type, EVs released by infected cells have differences in cargo, as they carry viral and host factors determined by the virus. The tetraspanin CD63 negatively impacts the infection, as demonstrated by CD63-knockdown and overexpression assays. A proposed mechanism involves the activation of antiviral responses in cells receiving CD63-positive EVs released by infected cells. Overall, HSV-1 causes major alterations in EVs that could contribute to HSV-1 persistence and pathogenesis. (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 Density gradient (Differential) (ultra)centrifugation Dialysis Ultrafiltration after density gradient Ultrafiltration Filtration Protein markers EV: TSG101/ CD63/ CD81/ Alix/ B-actin/ Flotillin2/ ICP0 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Embryonic lung fibroblast hTERT transformed cells Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Orientation Top-down Rotor type SW 41 Ti Speed (g) 250000 Duration (min) 120 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 30 Membrane type NS Other Name other separation method Dialysis Other Name other separation method Ultrafiltration after density gradient Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ B-actin/ Flotillin2/ TSG101/ Alix/ CD81 Not detected EV-associated proteins ICP0 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-400 EV concentration Yes

	EV210466	2/3	Homo sapiens	Embryonic lung fibroblast hTERT transformed cells	DG (d)(U)C Dialysis UF after density gradient UF Filtration	Dogrammatzis C	2019	38%
Study summary Full title Biogenesis of Extracellular Vesicles during Herpes Simplex Virus 1 Infection: Role of the CD63 Tetraspanin. All authors Dogrammatzis C, Deschamps T, Kalamvoki M Journal J Virol Abstract Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The vir (show more...)Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The virus primarily infects mucoepithelial cells and establishes latent reservoirs in neurons in sensory ganglia. Frequent reactivation has been linked to severe diseases, especially in immunocompromised individuals. Earlier, we reported that viral and host factors are packaged in extracellular vesicles (EVs) and delivered to uninfected cells, where they activate antiviral responses and restrict virus infection. Here, we interrogated the effect of HSV-1 infection on EV biogenesis. We found that HSV-1 infection causes a decrease in the amount of intracellular CD63 protein with a concomitant increase in extracellular CD63. This observation correlates with our previous finding that infected cells release more CD63-positive EVs than uninfected cells. The stimulation of CD63 exocytosis requires virus replication. CD63 is a member of the tetraspanin family of proteins that traffics between the plasma membrane and endosomal compartments and has a role in sorting cargo into the EVs. Previously, we reported that in cells depleted of CD63, HSV-1 virus yields increased, and here we provide data showing that in cells overexpressing CD63, HSV-1 virus yields decreased. Taken together, our data indicate that CD63 negatively impacts HSV-1 infection and that the CD63-positive EVs could control the dissemination of the virus in the host. Perhaps EV release by HSV-1-infected cells is a mechanism that controls virus dissemination. Intercellular communication, especially in neurons, largely relies on EVs, and modulation of EVs is known to impact physiological processes. Here, we present evidence that HSV-1 infection causes major alterations in the biogenesis of EVs, including an increase in their number and an increase in the CD63-positive population of EVs. These alterations result in an enrichment of the milieu of infection with EVs carrying signatures from infected cells. In addition to changes in the origin and type, EVs released by infected cells have differences in cargo, as they carry viral and host factors determined by the virus. The tetraspanin CD63 negatively impacts the infection, as demonstrated by CD63-knockdown and overexpression assays. A proposed mechanism involves the activation of antiviral responses in cells receiving CD63-positive EVs released by infected cells. Overall, HSV-1 causes major alterations in EVs that could contribute to HSV-1 persistence and pathogenesis. (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 HSV-1 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Dialysis Ultrafiltration after density gradient Ultrafiltration Filtration Protein markers EV: TSG101/ CD81/ Alix/ B-actin/ Flotillin2/ ICP0 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Embryonic lung fibroblast hTERT transformed cells Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Orientation Top-down Rotor type SW 41 Ti Speed (g) 250000 Duration (min) 120 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 30 Membrane type NS Other Name other separation method Dialysis Other Name other separation method Ultrafiltration after density gradient Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin2/ B-actin/ ICP0/ TSG101/ Alix/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-400 EV concentration Yes

	EV210466	3/3	Homo sapiens	Embryonic lung fibroblast hTERT transformed cells	DG (d)(U)C Dialysis UF after density gradient UF Filtration	Dogrammatzis C	2019	38%
Study summary Full title Biogenesis of Extracellular Vesicles during Herpes Simplex Virus 1 Infection: Role of the CD63 Tetraspanin. All authors Dogrammatzis C, Deschamps T, Kalamvoki M Journal J Virol Abstract Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The vir (show more...)Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The virus primarily infects mucoepithelial cells and establishes latent reservoirs in neurons in sensory ganglia. Frequent reactivation has been linked to severe diseases, especially in immunocompromised individuals. Earlier, we reported that viral and host factors are packaged in extracellular vesicles (EVs) and delivered to uninfected cells, where they activate antiviral responses and restrict virus infection. Here, we interrogated the effect of HSV-1 infection on EV biogenesis. We found that HSV-1 infection causes a decrease in the amount of intracellular CD63 protein with a concomitant increase in extracellular CD63. This observation correlates with our previous finding that infected cells release more CD63-positive EVs than uninfected cells. The stimulation of CD63 exocytosis requires virus replication. CD63 is a member of the tetraspanin family of proteins that traffics between the plasma membrane and endosomal compartments and has a role in sorting cargo into the EVs. Previously, we reported that in cells depleted of CD63, HSV-1 virus yields increased, and here we provide data showing that in cells overexpressing CD63, HSV-1 virus yields decreased. Taken together, our data indicate that CD63 negatively impacts HSV-1 infection and that the CD63-positive EVs could control the dissemination of the virus in the host. Perhaps EV release by HSV-1-infected cells is a mechanism that controls virus dissemination. Intercellular communication, especially in neurons, largely relies on EVs, and modulation of EVs is known to impact physiological processes. Here, we present evidence that HSV-1 infection causes major alterations in the biogenesis of EVs, including an increase in their number and an increase in the CD63-positive population of EVs. These alterations result in an enrichment of the milieu of infection with EVs carrying signatures from infected cells. In addition to changes in the origin and type, EVs released by infected cells have differences in cargo, as they carry viral and host factors determined by the virus. The tetraspanin CD63 negatively impacts the infection, as demonstrated by CD63-knockdown and overexpression assays. A proposed mechanism involves the activation of antiviral responses in cells receiving CD63-positive EVs released by infected cells. Overall, HSV-1 causes major alterations in EVs that could contribute to HSV-1 persistence and pathogenesis. (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 ICP8 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Dialysis Ultrafiltration after density gradient Ultrafiltration Filtration Protein markers EV: CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Embryonic lung fibroblast hTERT transformed cells Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Orientation Top-down Rotor type SW 41 Ti Speed (g) 250000 Duration (min) 120 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 30 Membrane type NS Other Name other separation method Dialysis Other Name other separation method Ultrafiltration after density gradient Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-400 EV concentration Yes

	EV210019	1/1	Homo sapiens	Primary adipose-derived mesenchymal stem cells	(d)(U)C	Mou, Shan	2019	38%
Study summary Full title Extracellular Vesicles from Human Adipose-Derived Stem Cells for the Improvement of Angiogenesis and Fat-Grafting Application All authors Shan Mou, Muran Zhou, Yuan Li, Jiecong Wang, Quan Yuan, Peng Xiao, Jiaming Sun, Zhenxing Wang Journal Plast Reconstr Surg. Abstract Background: The efficacy of autologous fat transplantation is reduced by fat absorption and fibrosis (show more...)Background: The efficacy of autologous fat transplantation is reduced by fat absorption and fibrosis that are closely related to unsatisfactory vascularization. Extracellular vesicles are key components of the cell secretome, which can mirror the functional and molecular characteristics of their parental cells. Growing evidence has revealed that adipose-derived mesenchymal stem cells have the ability to enhance vascularization, which is partly ascribed to extracellular vesicles. The authors evaluated whether adipose-derived mesenchymal stem cell-derived extracellular vesicles improved vascularization of fat grafts and increased their retention rate. Methods: To test the angiogenesis ability of adipose-derived mesenchymal stem cell-derived extracellular vesicles, they were isolated from the supernatant of cultured human adipose-derived mesenchymal stem cells and incubated with human umbilical vein endothelial cells in vitro. Then, the vesicles were co-transplanted with fat into nude mice subcutaneously. Three months after transplantation, the retention rate and inflammatory reaction of the grafts were analyzed by histologic assay. Results: The experimental group could significantly promote migration and tube formation at the concentration of 20 μg/ml. At 3 months after transplantation, the volume of the experimental group (0.12 ± 0.03 mm) was larger compared with the blank group (0.05 ± 0.01 mm). Histology and immunohistology results demonstrated significantly fewer cysts and vacuoles, less fibrosis, and more neovessels in the extracelluar vesicle group. Conclusions: The authors co-transplanted adipose-derived mesenchymal stem cell-derived extracellular vesicles with fat into a nude mouse model and found that the vesicles improved volume retention by enhancing vascularization and regulating the inflammatory response. (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 (Differential) (ultra)centrifugation Protein markers EV: CD81/ b-actin/ VEGF/ 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 mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 16000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ b-actin/ VEGF/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 100-1000 Used for determining EV concentration? Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210018	1/1	Mus musculus	Primary adipose-derived mesenchymal stem cells	(d)(U)C Filtration UF	Chen, Bin	2019	38%
Study summary Full title Exosomes Are Comparable to Source Adipose Stem Cells in Fat Graft Retention with Up-Regulating Early Inflammation and Angiogenesis All authors Bin Chen, Junrong Cai, Yating Wei, Zhaohua Jiang, Haley E Desjardins, Alexandra E Adams, Shengli Li, Huang-Kai Kao, Lifei Guo Journal Plast Reconstr Surg. Abstract Background: Exosomes derived from mesenchymal stem cells possess functional properties similar to th (show more...)Background: Exosomes derived from mesenchymal stem cells possess functional properties similar to those of their parent cells, suggesting that they could play a pivotal role in tissue repair and regeneration. Methods: Using lipotransfer as a surrogate, exosomes were isolated from mouse adipose-derived stem cell-conditioned medium and characterized. Minced fat tissue mixed with exosomes, source cells (cell-assisted lipotransfer), or saline was implanted subcutaneously in the lower back of C57/BL mice bilaterally (n = 16 each). Transferred fat tissues were harvested and analyzed at 3 and 10 weeks. Results: At 3 and 10 weeks after the transfer, fat grafts in groups of exosomes and cell-assisted lipotransfer showed better fat integrity, fewer oil cysts, and reduced fibrosis. At week 10, graft retention rates in cell-assisted lipotransfer (50.9 ± 2.4 percent; p = 0.03) and exosome groups (56.4 ± 1.6 percent; p < 0.001) were significantly higher than in the saline group (40.7 ± 4.7 percent). Further investigations of macrophage infiltration, inflammatory factors, angiogenic factors, adipogenic factors, and extracellular matrix revealed that those exosomes promoted angiogenesis and up-regulated early inflammation, whereas during mid to late stages of fat grafting, they exerted a proadipogenic effect and also increased collagen synthesis level similarly to their source cells. Conclusions: The adipose-derived stem cell-derived exosomes demonstrated effects comparable to those of their source cells in achieving improved graft retention by up-regulating early inflammation and augmenting angiogenesis. These features may enable exosomes to be an attractive cell-free alternative in therapeutic regenerative medicine. (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 Filtration Ultrafiltration Protein markers EV: CD9 non-EV: b-actin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Primary adipose-derived mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm0.2µm > x > 0.1µm Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9 Detected contaminants b-actin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 123 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200167	8/9	Mus musculus	C2C12	(d)(U)C	Ghamloush, Farah	2019	38%
Study summary Full title The PAX3-FOXO1 oncogene alters exosome miRNA content and leads to paracrine effects mediated by exosomal miR-486 All authors Farah Ghamloush, Sandra E Ghayad, Ghina Rammal, Assil Fahs, Abeer J Ayoub, Zeina Merabi, Mohamad Harajly, Hassan Zalzali, Raya Saab 5 Journal Sci Rep Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARM (show more...)Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARMS) is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 that drives oncogenic cellular properties. Exosomes are small, secreted vesicles that affect paracrine signaling. We show that PAX3-FOXO1 transcript alters exosome content of C2C12 myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells. Microarray analysis revealed alteration in miRNA content of exosomes, affecting cellular networks involved in cell metabolism, growth signaling, and cellular invasion. Overexpression and knockdown studies showed that miR-486-5p is an effector of PAX3-FOXO1, and mediates its paracrine effects in exosomes, including promoting recipient cell migration, invasion, and colony formation. Analysis of human RMS cells showed miR-486-5p is enriched in both cells and exosomes, and to a higher extent in ARMS subtypes. Analysis of human serum samples showed that miR-486-5p is enriched in exosomes of patients with RMS, and follow-up after chemotherapy showed decrease to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker. (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 Protein markers EV: TSG101/ GAPDH/ HSC70/ PAX3-FOXO1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biomarker Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells C2C12 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins HSC70/ GAPDH/ TSG101 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM Image type Wide-field Report size (nm) 40-100

	EV200167	9/9	Mus musculus	C2C12	(d)(U)C	Ghamloush, Farah	2019	38%
Study summary Full title The PAX3-FOXO1 oncogene alters exosome miRNA content and leads to paracrine effects mediated by exosomal miR-486 All authors Farah Ghamloush, Sandra E Ghayad, Ghina Rammal, Assil Fahs, Abeer J Ayoub, Zeina Merabi, Mohamad Harajly, Hassan Zalzali, Raya Saab 5 Journal Sci Rep Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARM (show more...)Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARMS) is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 that drives oncogenic cellular properties. Exosomes are small, secreted vesicles that affect paracrine signaling. We show that PAX3-FOXO1 transcript alters exosome content of C2C12 myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells. Microarray analysis revealed alteration in miRNA content of exosomes, affecting cellular networks involved in cell metabolism, growth signaling, and cellular invasion. Overexpression and knockdown studies showed that miR-486-5p is an effector of PAX3-FOXO1, and mediates its paracrine effects in exosomes, including promoting recipient cell migration, invasion, and colony formation. Analysis of human RMS cells showed miR-486-5p is enriched in both cells and exosomes, and to a higher extent in ARMS subtypes. Analysis of human serum samples showed that miR-486-5p is enriched in exosomes of patients with RMS, and follow-up after chemotherapy showed decrease to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker. (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 PAX3-FOXO1 transfected Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: TSG101/ GAPDH/ HSC70/ PAX3-FOXO1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biomarker Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells C2C12 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins HSC70/ GAPDH/ TSG101 Not detected EV-associated proteins PAX3-FOXO1 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM Image type Wide-field Report size (nm) 40-100

	EV200165	8/8	Homo sapiens	Blood plasma	ExoRNeasy serum/plasma Midi Kit (Qiagen)	Ma, Jing	2019	38%
Study summary Full title Exosomal hsa-miR199a-3p Promotes Proliferation and Migration in Neuroblastoma All authors Jing Ma, Min Xu, Minzhi Yin, Jie Hong, Haoyan Chen, Yijin Gao, Chenjie Xie, Nan Shen, Song Gu, Xi Mo Journal Front Oncol Abstract Neuroblastoma (NB) is the most common pediatric extra-cranial solid tumor with heterogeneous charact (show more...)Neuroblastoma (NB) is the most common pediatric extra-cranial solid tumor with heterogeneous characteristics, and the prognosis of patients with high-risk NB is usually poor. Discovery of novel biomarkers for early detection and investigation of the underlying mechanisms governing invasion and metastasis of NB are urgently needed. Recently, exosomal microRNAs (miRNAs) have been shown to play vital regulatory or communication roles in the process of various types of cancers. However, the roles and mechanisms of exosomal miRNAs in NB remain unknown. Thus, the present study aims to investigate the detailed functions of tumor-derived exosomal miRNAs in progression and migration of NB in vivo and in vitro. By examining different exosomal miRNA expression profiles in the plasma of NB patients, we identified that the expression of hsa-miR199a-3p from exosomes was significantly upregulated, which was correlated with the severity of NB patients. Furthermore, we confirmed that exosomal hsa-miR199a-3p could facilitate proliferation and migration of NB via regulating NEDD4 expression. In summary, our data, for the first time, revealed that exosomal hsa-miR199a-3p could promote tumor proliferation and migration via decreasing NEDD4 expression in NB, suggesting that exosomal hsa-miR199a-3p may be applicated as a fast, easy, and non-invasive detection biomarker and contribute to the development of novel therapeutic strategies for NB in the future. (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 Neuroblastoma 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 ExoRNeasy serum/plasma Midi Kit (Qiagen) Protein markers EV: CD81/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;ExoRNeasy serum/plasma Midi Kit (Qiagen) Other Name other separation method ExoRNeasy serum/plasma Midi Kit (Qiagen) Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins CD63 Not detected contaminants Calnexin Flow cytometry Type of Flow cytometry Accuri C6 flow cytometer (BD Instruments) Calibration bead size Not specified Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 79.42 EM EM-type Transmission-EM Image type Close-up

	EV200048	2/2	Gallus gallus	semen	IAF (d)(U)C ExoQuick Filtration	Sheng Chen	2019	38%
Study summary Full title MicroRNA expression profile in extracellular vesicles derived from ALV-J infected chicken semen All authors Sheng Chen, Liqin Liao, Qiqi Zhao, Xinheng Zhang, Hongxin Li, Wencheng Lin, Feng Chen, Qingmei Xie Journal Virus Res Abstract MicroRNAs(miRNAs) have been reported to regulate gene expression in many processes. MiRNA in extrace (show more...)MicroRNAs(miRNAs) have been reported to regulate gene expression in many processes. MiRNA in extracellular vesicles (EVs) also have been widely investigated, while there is no studies of miRNAs in seminal EVs. Subgroup J of Avian leukosis virus (ALV-J) can be transmitted vertically, but the mechanism of it is not clear enough. This study was to examine the miRNA expression profile in seminal EVs and inquire into the relation between it and the vertical transmission by performing gene ontology (GO) and pathway enrichment analysis. Here, we first isolated and characterized seminal EVs by Nanoparticle Tracking Analysis、Western Blot and Transmission electron microscopy experiments. By deep sequencing of each EVs miRNA library, 9 typical differentially expressed miRNA, including 6 up-regulated and 3 down-regulated, were identified. Gene target prediction, GO annotation and KEGG pathway enrichment analysis showed possible function associated with these miRNAs. Overall, these findings will increase our understanding of the content and composition of miRNA in seminal EVs and provide new insights into the important role of the seminal EVs miRNAs regulation in ALV-J transmission. (hide) EV-METRIC 38% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type semen Sample origin ALV-J infected Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Immunoaffinity capture (non-commercial) (d)(U)C Commercial method Filtration Protein markers EV: TSG101/ CD63/ CD81/ GRP78/ HSP70/ CD9 non-EV: None Proteomics no Show all info Study aim NA Sample Species Gallus gallus Sample Type semen Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ CD81 Not detected EV-associated proteins HSP70/ CD81/ GRP78/ TSG101/ CD63/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins HSP70/ CD63/ CD81/ CD9/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 127 EV concentration Yes Particle yield particles/ml;Yes, other: 8580000000

	EV200045	5/5	Homo sapiens	UMSCC47	(d)(U)C SEC (non-commercial) Filtration UF	Ludwig Nils	2019	38%
Study summary Full title Isolation and Analysis of Tumor-Derived Exosomes All authors Ludwig N, Hong CS, Ludwig S, Azambuja JH, Sharma P, Theodoraki MN, Whiteside TL. Journal Current Protocols in Immunology Abstract A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presen (show more...)A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presented. Tumor-derived exosomes (TEX) are defined as a subset of extracellular vesicles (EVs) sized at 30 to 150 nm and originating from multivesicular bodies (MVBs). The method utilizes size exclusion chromatography (SEC) for recovery of exosomes from cell-line supernatants or cancer patients' plasma. The recovered exosomes are morphologically intact, aggregate-free, and functionally competent. Their molecular content parallels that of the parent tumor cells and they carry various immunoregulatory ligands known to modulate functions of immune cells. All exosomes isolated from tumor cell lines are TEX, while those isolated from plasma of cancer patients have to be fractionated into TEX and non-TEX. Mini-SEC allows for exosome isolation and recovery in quantities sufficient for molecular profiling, functional studies, and, in the case of plasma, further fractionation into TEX and non-TEX. The mini-SEC method can also be used for comparative studies of the exosome content in serial specimens of cancer patients' body fluids. (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 (d)(U)C SEC (non-commercial) Filtration UF Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UMSCC47 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 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method SEC (non-commercial) PMID previous EV protein analysis 30042174 + 30370252 Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins PMID 30042174 / PMID 30370252 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV190080	2/3	Homo sapiens	A2780	DG	Zaborowski MP	2019	38%
Study summary Full title Effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women. All authors Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, Zappulli V, Maas SLN, Allen RM, Rumde P, György B, Aufiero M, Schweiger MW, Lai CP, Weissleder R, Lee H, Vickers KC, Tannous BA, Breakefield XO. Journal Sci Rep Abstract Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter (show more...)Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models. (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 DG Protein markers EV: CD63 non-EV: Proteomics no EV density (g/ml) Not specified Show all info Study aim New methodological development/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A2780 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 8% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 200620 Duration (min) 38 Fraction volume (mL) 0.35 Fraction processing None Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Other 1 https://www.ncbi.nlm.nih.gov/pubmed/30943406 Characterization: Lipid analysis No Other particle analysis name(1) Report type EV-concentration

	EV190012	1/3	Mus musculus	4T1	DC (d)(U)C Filtration qEV UF	Vu LT	2019	38%
Study summary Full title Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b. All authors Vu LT, Peng B, Zhang DX, Ma V, Mathey-Andrews CA, Lam CK, Kiomourtzis T, Jin J, McReynolds L, Huang L, Grimson A, Cho WC, Lieberman J, Le MT. Journal J Extracell Vesicles Abstract Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions (show more...)Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts. (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 DC (d)(U)C Filtration qEV UF Protein markers EV: TSG101/ Alix/ CD63 non-EV: Beta-Actin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) Not spec Membrane type Regenerated cellulose Commercial kit qEV Density cushion Density medium Sucrose Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ Alix Not detected contaminants Beta-Actin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD63 Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR; RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 50 U Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV190008	2/2	Homo sapiens	HEK 293	(d)(U)C ExoQuick Filtration	Duong N	2019	38%
Study summary Full title Decoy exosomes as a novel biologic reagent to antagonize inflammation. All authors Duong N, Curley K, Brown A, Campanelli A, Do MA, Levy D, Tantry A, Marriott G, Lu B. Journal Nanomedicine Abstract Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to (show more...)Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα. (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 TNFalpha-CD63-GFP overexpressing 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 ExoQuick Filtration Protein markers EV: / TSG101/ CD63/ CD81/ Alix/ ICAM/ Flotillin1/ HSP70 non-EV: / GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK 293 EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Other;UV-Vis Microvolume Spectrophotometer Western Blot Antibody details provided? No Detected EV-associated proteins Not detected contaminants ELISA Antibody details provided? No Detected EV-associated proteins Flow cytometry Type of Flow cytometry Calibration bead size Antibody details provided? No Detected EV-associated proteins Other 1 Dot blot Detected EV-associated proteins Flotillin1/ Alix/ CD63/ ICAM/ TSG101/ HSP70/ CD81 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 46

	EV180025	1/3	Homo sapiens	Blood plasma	(d)(U)C SEC	Gámez-Valero A	2019	37%
Study summary Full title Comprehensive proteomic profiling of plasma-derived Extracellular Vesicles from dementia with Lewy Bodies patients. All authors Gámez-Valero A, Campdelacreu J, Reñé R, Beyer K, Borràs FE. Journal Sci Rep Abstract Proteins and nucleic acids contained in extracellular vesicles (EVs) are considered a feasible sourc (show more...)Proteins and nucleic acids contained in extracellular vesicles (EVs) are considered a feasible source of putative biomarkers for physiological and pathological conditions. Within the nervous system, not only neurons but also other brain cells are able to produce EVs, which have been involved in their physiological processes and also in the development and course of several neurodegenerative diseases. Among these, dementia with Lewy bodies (DLB) is the second cause of dementia worldwide, though most cases are missed or misdiagnosed as Alzheimer's disease (AD) due to the important clinical and pathological overlap between both diseases. In an attempt to find reliable biomarkers for DLB diagnosis, our group characterized the proteome of plasma-derived EVs from DLB patients compared to aged-matched healthy controls (HCs) using two different proteomic LC-MS/MS approaches. Remarkably, we found that gelsolin and butyrylcholinesterase were differentially identified between DLB and HCs. Further validation of these results using conventional ELISA techniques, and including an additional group of AD patients, pointed to decreased levels of gelsolin in plasma-EVs from DLB compared to HCs and to AD samples. Thus, gelsolin may be considered a possible biomarker for the differentiation between DLB and AD. (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 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 SEC Protein markers EV: CD81/ CD63/ CD9/ CD5L non-EV: None Proteomics yes 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 Size-exclusion chromatography Total column volume (mL) 15 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA ELISA Antibody details provided? No Lysis buffer provided? Yes Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV170040	2/2	Rattus norvegicus	Serum	Filtration PEG precipitation	Voglstaetter, Maren	2019	37%
Study summary Full title Tspan8 is expressed in breast cancer and regulates E‐cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles All authors Maren Voglstaetter, Andreas R Thomsen, Jerome Nouvel, Arend Koch, Paul Jank, Elena Grueso Navarro, Tanja Gainey‐Schleicher, Richa Khanduri, Andrea Groß, Florian Rossner, Carina Blaue, Clemens M Franz, Marina Veil, Gerhard Puetz, Andreas Hippe, Jochen Dindorf, Jubin Kashef, Wilko Thiele, Bernhard Homey, Celine Greco, Claude Boucheix, Andreas Baur, Thalia Erbes, Cornelius F Waller, Marie Follo, Ghamartaj Hossein, Christine Sers, Jonathan Sleeman, Irina Nazarenko Journal J Pathol Abstract Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus (show more...)Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. (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 Serum Sample origin Breast 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 Filtration PEG precipitation Protein markers EV: TSG101/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Serum Separation Method Filtration steps 0.22µm or 0.2µm Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 10 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type size distribution by intensty and by mass are reported Reported size (nm) 10-100 nm; 1000-9000 nm NTA Report type Size range/distribution Reported size (nm) 100 EV concentration Yes Particle yield 2.00E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up

	EV210068	3/6	Homo sapiens	Blood plasma	(d)(U)C	Gill, Manjot	2019	34%
Study summary Full title Placental Syncytiotrophoblast-Derived Extracellular Vesicles Carry Active NEP (Neprilysin) and Are Increased in Preeclampsia All authors Manjot Gill, Carolina Motta-Mejia, Neva Kandzija, William Cooke, Wei Zhang, Ana Sofia Cerdeira, Claire Bastie, Christopher Redman, Manu Vatish Journal Hypertension Abstract NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a var (show more...)NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension-a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas ( P<0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan ( P<0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry ( P<0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia ( P<0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease. (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 Pregnant, placental blood Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: NEP/ PLAP non-EV: None Proteomics no Show all info Study aim Function/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 Yes Pelleting: time(min) 30 Pelleting: rotor type TST28.39 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type TST28.39 Wash: speed (g) 150000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins NEP/ PLAP Detected EV-associated proteins NEP/ PLAP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210068	4/6	Homo sapiens	Blood plasma	(d)(U)C Filtration	Gill, Manjot	2019	34%
Study summary Full title Placental Syncytiotrophoblast-Derived Extracellular Vesicles Carry Active NEP (Neprilysin) and Are Increased in Preeclampsia All authors Manjot Gill, Carolina Motta-Mejia, Neva Kandzija, William Cooke, Wei Zhang, Ana Sofia Cerdeira, Claire Bastie, Christopher Redman, Manu Vatish Journal Hypertension Abstract NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a var (show more...)NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension-a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas ( P<0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan ( P<0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry ( P<0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia ( P<0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease. (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 Pregnant, placental blood Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: NEP/ PLAP non-EV: None Proteomics no Show all info Study aim Function/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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TST28.39 Pelleting: speed (g) 150000 Wash: volume per pellet (ml) Not specified Wash: time (min) 30 Wash: Rotor Type TST28.39 Wash: speed (g) 10000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins NEP/ PLAP Detected EV-associated proteins NEP/ PLAP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210068	5/6	Homo sapiens	Blood plasma	(d)(U)C	Gill, Manjot	2019	34%
Study summary Full title Placental Syncytiotrophoblast-Derived Extracellular Vesicles Carry Active NEP (Neprilysin) and Are Increased in Preeclampsia All authors Manjot Gill, Carolina Motta-Mejia, Neva Kandzija, William Cooke, Wei Zhang, Ana Sofia Cerdeira, Claire Bastie, Christopher Redman, Manu Vatish Journal Hypertension Abstract NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a var (show more...)NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension-a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas ( P<0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan ( P<0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry ( P<0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia ( P<0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease. (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 Pregnant, preeclampsia, placental blood Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: NEP/ PLAP non-EV: None Proteomics no Show all info Study aim Function/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 Yes Pelleting: time(min) 30 Pelleting: rotor type TST28.39 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type TST28.39 Wash: speed (g) 150000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins NEP/ PLAP Detected EV-associated proteins NEP/ PLAP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210068	6/6	Homo sapiens	Blood plasma	(d)(U)C Filtration	Gill, Manjot	2019	34%
Study summary Full title Placental Syncytiotrophoblast-Derived Extracellular Vesicles Carry Active NEP (Neprilysin) and Are Increased in Preeclampsia All authors Manjot Gill, Carolina Motta-Mejia, Neva Kandzija, William Cooke, Wei Zhang, Ana Sofia Cerdeira, Claire Bastie, Christopher Redman, Manu Vatish Journal Hypertension Abstract NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a var (show more...)NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension-a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas ( P<0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan ( P<0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry ( P<0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia ( P<0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease. (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 Pregnant, preeclampsia, placental blood Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: NEP/ PLAP non-EV: None Proteomics no Show all info Study aim Function/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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TST28.39 Pelleting: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins NEP/ PLAP Detected EV-associated proteins NEP/ PLAP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210028	1/2	Homo sapiens	Primary adipose-derived stem cells	(d)(U)C Filtration	Han, Yudi	2019	34%
Study summary Full title Exosomes from hypoxia-treated human adipose-derived mesenchymal stem cells enhance angiogenesis through VEGF/VEGF-R All authors Yudi Han, Jing Ren, Yun Bai, Xuetao Pei, Yan Han Journal Int J Biochem Cell Biol Abstract Background: We previously reported that co-transplantation of exosomes from hypoxia-preconditioned a (show more...)Background: We previously reported that co-transplantation of exosomes from hypoxia-preconditioned adipose mesenchymal stem cells (ADSCs) improves the neoangiogenesis and survival of the grafted tissue. This study aimed to investigate the molecular mechanism of this protective effect. Methods: Exosomes were collected from normoxia-treated (nADSC-Exo) or hypoxia--treated (hypADSC-Exo) human ADSCs, and their pro-angiogenic capacity was evaluated in human umbilical vein endothelial cells (HUVECs) and a nude mouse model of subcutaneous fat grafting. Protein array was used to compare the exosome-derived proteins between nADSC-Exo and hypADSC-Exo. Results: Compared with the nADSC-Exo group and untreated control, hypADSC-Exo treatment significantly promoted proliferation, migration and tube-formation capability of HUVECs. Protein array revealed that the levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and their receptors (VEGF-R2, VEGF-R3), and monocyte chemoattractant protein 2 (MCP-2), monocyte chemoattractant protein 4 (MCP-4) were significantly higher in the hypADSC-Exo than in the nADSC-Exo. In the nude mice model of fat grafting, immunofluorescence of CD31 showed that hypADSC-Exo dramatically improved neovascularization around the graft. Furthermore, compared with nADSC-Exo and control groups, cotransplantation of hypADSC-Exo significantly increased the protein expression of EGF, FGF, VEGF/VEGF-R, angiopoietin-1(Ang-1) and tyrosine kinase with immunoglobulin-like and EGF-like domains 1(Tie-1, an angiopoietin receptor) in the grafted tissue at 30 days after transplantation. Immunohistochemical analysis demonstrated that hypADSC-Exo treatment significantly increased VEGF-R expression in the grafted tissue. Conclusions: Exosomes from hypoxia-treated human ADSCs possess a higher capacity to enhance angiogenesis in fat grafting, at least partially, via regulating VEGF/VEGF-R signaling. (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/ VEGF-A, VEGF-R2/ CD63/ CD34/ CD90/ CD34, CD90/ CD9 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 adipose-derived stem cells 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) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD34, CD90/ VEGF-A, VEGF-R2/ TSG101/ GAPDH Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins TSG101/ CD34/ CD90/ CD9/ CD63 Proteomics database No Other 1 Human growth factor antibody array Detected EV-associated proteins GSH-ANG-3, RayBiotech, USA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 75 EV concentration Yes Particle yield NA NA EM EM-type Transmission-EM Image type Wide-field

	EV200143	2/2	Homo sapiens	Blood plasma	(d)(U)C Filtration	Ramkumar, Menon	2019	34%
Study summary Full title Circulating Exosomal miRNA Profile During Term and Preterm Birth Pregnancies: A Longitudinal Study All authors Ramkumar Menon, Chirantan Debnath, Andrew Lai, Dominic Guanzon, Shinjini Bhatnagar, Pallavi K Kshetrapal, Samantha Sheller-Miller, Carlos Salomon, Garbhini Study Team Journal Endocrinology Abstract Despite decades of research in the field of human reproduction, the mechanisms responsible for human (show more...)Despite decades of research in the field of human reproduction, the mechanisms responsible for human parturition still remain elusive. The objective of this study was to describe the changes in the exosomal miRNA concentrations circulating in the maternal plasma between mothers delivering term and preterm neonates, across gestation using a longitudinal study design. This descriptive study identifies the miRNA content in exosomes present in maternal plasma of term and preterm birth (PTB) (n = 20 and n = 10 per each gestational period, respectively) across gestation (i.e., first, second, and third trimesters and at the time of delivery). Changes in exosomal miRNA signature in maternal plasma during term and preterm gestation were determined using the NextSeq 500 high-output 75 cycles sequencing platform. A total of 167 and 153 miRNAs were found to significantly change (P < 0.05) as a function of the gestational age across term and PTB pregnancies, respectively. Interestingly, a comparison analysis between the exosomal miRNA profile between term and PTB reveals a total of 173 miRNAs that significantly change (P < 0.05) across gestation. Specific trends of changes (i.e., increase, decrease, and both) as a function of the gestational age were also identified. The bioinformatics analyses establish that the differences in the miRNA profile are targeting signaling pathways associated with TGF-β signaling, p53, and glucocorticoid receptor signaling, respectively. These data suggest that the miRNA content of circulating exosomes in maternal blood might represent a biomolecular fingerprint of the progression of pregnancy. (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 Pre-term birth 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: Grp94 Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants Grp94 Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-120nm

	EV230017	1/3	Leptopilina boulardi	Wasp venom apparatus	(d)(U)C DG	Wan B	2019	33%
Study summary Full title Venom Atypical Extracellular Vesicles as Interspecies Vehicles of Virulence Factors Involved in Host Specificity: The Case of a Parasitoid Wasp. All authors Wan B, Goguet E, Ravallec M, Pierre O, Lemauf S, Volkoff AN, Gatti JL, Poirié M Journal Front Immunol Abstract Endoparasitoid wasps, which lay eggs inside the bodies of other insects, use various strategies to p (show more...)Endoparasitoid wasps, which lay eggs inside the bodies of other insects, use various strategies to protect their offspring from the host immune response. The hymenopteran species of the genus , parasites of , rely on the injection of a venom which contains proteins and peculiar vesicles (hereafter venosomes). We show here that the injection of purified venosomes is sufficient to impair the function of the lamellocytes, a hemocyte type specialized in the defense against wasp eggs, and thus the parasitic success of the wasp. These venosomes seem to have a unique extracellular biogenesis in the wasp venom apparatus where they acquire specific secreted proteins/virulence factors and act as a transport system to deliver these compounds into host lamellocytes. The level of venosomes entry into lamellocytes of different species was correlated with the rate of parasitism success of the wasp, suggesting that this venosome-cell interaction may represent a new evolutionary level of host-parasitoid specificity. (hide) EV-METRIC 33% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Wasp venom apparatus Sample origin Strain ISy Focus vesicles Venosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: LbGAP/ LbGAP2/ LbSPN non-EV: None Proteomics no Show all info Study aim Function Sample Species Leptopilina boulardi Sample Type Wasp venom apparatus Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: speed (g) 15000 Wash: time (min) 15 Wash: speed (g) 15000 Density gradient Only used for validation of main results Yes Lowest density fraction 10% Highest density fraction 50% Orientation Top-down Rotor type MLA-50 Speed (g) 35000 Duration (min) 60 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins LbGAP/ LbGAP2/ LbSPN Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 100-300

	EV220285	1/1	Homo sapiens	Menstrual blood-derived mesenchymal stem cells	(d)(U)C	Dalirfardouei R	2019	33%
Study summary Full title Promising effects of exosomes isolated from menstrual blood-derived mesenchymal stem cell on wound-healing process in diabetic mouse model. All authors Dalirfardouei R, Jamialahmadi K, Jafarian AH, Mahdipour E Journal J Tissue Eng Regen Med Abstract Wound healing is a complicated process that contains a number of overlapping and consecutive phases, (show more...)Wound healing is a complicated process that contains a number of overlapping and consecutive phases, disruption in each of which can cause chronic nonhealing wounds. In the current study, we investigated the effects of exosomes as paracrine factors released from menstrual blood-derived mesenchymal stem cells (MenSCs) on wound-healing process in diabetic mice. The exosomes were isolated from MenSCs conditioned media using ultracentrifugation and were characterized by scanning electron microscope and western blotting assay. A full thickness excisional wound was created on the dorsal skin of each streptozotocin-induced diabetic mouse. The mice were divided into three groups as follows: phosphate buffered saline, exosomes, and MenSC groups. We found that MenSC-derived exosomes can resolve inflammation via induced M1-M2 macrophage polarization. It was observed that exosomes enhance neoangiogenesis through vascular endothelial growth factor A upregulation. Re-epithelialization accelerated in the exosome-treated mice, most likely through NF-κB p65 subunit upregulation and activation of the NF-κB signaling pathway. The results demonstrated that exosomes possibly cause less scar formation through decreased Col1:Col3 ratio. These notable results showed that the MenSC-derived exosomes effectively ameliorated cutaneous nonhealing wounds. We suggest that exosomes can be employed in regenerative medicine for skin repair in difficult-to-heal conditions such as diabetic foot ulcer. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: TSG101/ CD81 non-EV: calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Menstrual blood-derived mesenchymal 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method No Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins TSG101/ CD81 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM/ Atomic force microscopy Image type Close-up, Wide-field Report size (nm) 40-200 Report type Not Reported EV-concentration No

	EV220284	1/1	Mus musculus	Adipose-derived mesenchymal stem cells	(d)(U)C Filtration UF	Wang C	2019	33%
Study summary Full title Engineering Bioactive Self-Healing Antibacterial Exosomes Hydrogel for Promoting Chronic Diabetic Wound Healing and Complete Skin Regeneration. All authors Wang C, Wang M, Xu T, Zhang X, Lin C, Gao W, Xu H, Lei B, Mao C Journal Theranostics Abstract Chronic nonhealing diabetic wound therapy and complete skin regeneration remains a critical clinical (show more...)Chronic nonhealing diabetic wound therapy and complete skin regeneration remains a critical clinical challenge. The controlled release of bioactive factors from a multifunctional hydrogel was a promising strategy to repair chronic wounds. Herein, for the first time, we developed an injectable, self-healing and antibacterial polypeptide-based FHE hydrogel (F127/OHA-EPL) with stimuli-responsive adipose-derived mesenchymal stem cells exosomes (AMSCs-exo) release for synergistically enhancing chronic wound healing and complete skin regeneration. The materials characterization, antibacterial activity, stimulated cellular behavior and full-thickness diabetic wound healing ability of the hydrogels were performed and analyzed. The FHE hydrogel possessed multifunctional properties including fast self-healing process, shear-thinning injectable ability, efficient antibacterial activity, and long term pH-responsive bioactive exosomes release behavior. , the FHE@exosomes (FHE@exo) hydrogel significantly promoted the proliferation, migration and tube formation ability of human umbilical vein endothelial cells (HUVECs). , the FHE@exo hydrogel significantly enhanced the healing efficiency of diabetic full-thickness cutaneous wounds, characterized with enhanced wound closure rates, fast angiogenesis, re-epithelization and collagen deposition within the wound site. Moreover, the FHE@exo hydrogel displayed better healing outcomes than those of exosomes or FHE hydrogel alone, suggesting that the sustained release of exosomes and FHE hydrogel can synergistically facilitate diabetic wound healing. Skin appendages and less scar tissue also appeared in FHE@exo hydrogel treated wounds, indicating its potent ability to achieve complete skin regeneration. This work offers a new approach for repairing chronic wounds completely through a multifunctional hydrogel with controlled exosomes release. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Ultrafiltration Protein markers EV: Alix/ CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Adipose-derived mesenchymal stem cells Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 100 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ Alix/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 60-80 EM EM-type Transmission-EM Image type Close-up

	EV220280	1/2	Homo sapiens	Human umbilical vein endothelial cells	(d)(U)C Filtration	Zeng T	2019	33%
Study summary Full title Endothelial cell-derived small extracellular vesicles suppress cutaneous wound healing through regulating fibroblasts autophagy. All authors Zeng T, Wang X, Wang W, Feng Q, Lao G, Liang Y, Wang C, Zhou J, Chen Y, Liu J, Gao H, Lan B, Wu Y, Han Y, Liu Y, Chen H, Liu L, Yang C, Yan L, Ren M, Sun K Journal Clin Sci (Lond) Abstract Diabetic foot ulcer is a life-threatening clinical problem in diabetic patients. Endothelial cell-de (show more...)Diabetic foot ulcer is a life-threatening clinical problem in diabetic patients. Endothelial cell-derived small extracellular vesicles (sEVs) are important mediators of intercellular communication in the pathogenesis of several diseases. However, the exact mechanisms of wound healing mediated by endothelial cell-derived sEVs remain unclear. sEVs were isolated from human umbilical vein endothelial cells (HUVECs) pretreated with or without advanced glycation end products (AGEs). The roles of HUVEC-derived sEVs on the biological characteristics of skin fibroblasts were investigated both and We demonstrate that sEVs derived from AGEs-pretreated HUVECs (AGEs-sEVs) could inhibit collagen synthesis by activating autophagy of human skin fibroblasts. Additionally, treatment with AGEs-sEVs could delay the wound healing process in Sprague-Dawley (SD) rats. Further analysis indicated that miR-106b-5p was up-regulated in AGEs-sEVs and importantly, in exudate-derived sEVs from patients with diabetic foot ulcer. Consequently, sEV-mediated uptake of miR-106b-5p in recipient fibroblasts reduces expression of extracellular signal-regulated kinase 1/2 (ERK1/2), resulting in fibroblasts autophagy activation and subsequent collagen degradation. Collectively, our data demonstrate that miR-106b-5p could be enriched in AGEs-sEVs, then decreases collagen synthesis and delays cutaneous wound healing by triggering fibroblasts autophagy through reducing ERK1/2 expression. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD63/ CD9 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 Human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants Grp94 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ miRNA microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-200 EM EM-type Transmission-EM Image type Close-up

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