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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200042	1/2	Mus musculus	Bone marrow-derived macrophages (BMDMs)	DG (d)(U)C DC Filtration	Laura Bouchareychas	2020	75%
Study summary Full title Macrophage Exosomes Resolve Atherosclerosis by Regulating Hematopoiesis and Inflammation via MicroRNA Cargo All authors Laura Bouchareychas, Phat Duong, Sergio Covarrubias, Eric Alsop, Tuan Anh Phu, Allen Chung, Michael Gomes, David Wong, Bessie Meechoovet, Allyson Capili, Ryo Yamamoto, Hiromitsu Nakauchi, Michael T McManus, Susan Carpenter, Kendall Van Keuren-Jensen, Robert L Raffai Journal Cell Rep Abstract Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remai (show more...)Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery. (hide) EV-METRIC 75% (96th 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 DG (d)(U)C DC Filtration Protein markers EV: Alix/ Flotillin1/ CD9 non-EV: Calnexin/ GM130 Proteomics no EV density (g/ml) 1.09 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Bone marrow-derived macrophages (BMDMs) EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 5.00E+06 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 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Density cushion Density medium Iodixanol Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD9/ Alix Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type Other;RNase A/T1 Mix RNAse concentration 0.4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 75.66 EV concentration Yes Particle yield Yes, as number of particles per million cells 2.70E+09

	EV200042	2/2	Mus musculus	Immortalized bone marrow-derived macrophages (iBMDMs)	DG (d)(U)C DC Filtration	Laura Bouchareychas	2020	63%
Study summary Full title Macrophage Exosomes Resolve Atherosclerosis by Regulating Hematopoiesis and Inflammation via MicroRNA Cargo All authors Laura Bouchareychas, Phat Duong, Sergio Covarrubias, Eric Alsop, Tuan Anh Phu, Allen Chung, Michael Gomes, David Wong, Bessie Meechoovet, Allyson Capili, Ryo Yamamoto, Hiromitsu Nakauchi, Michael T McManus, Susan Carpenter, Kendall Van Keuren-Jensen, Robert L Raffai Journal Cell Rep Abstract Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remai (show more...)Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery. (hide) EV-METRIC 63% (93rd 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 genetically modified cell line Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C DC Filtration Protein markers EV: Alix/ Flotillin1 non-EV: Calnexin/ GM130 Proteomics no EV density (g/ml) 1.09 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Immortalized bone marrow-derived macrophages (iBMDMs) EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 3.50E+07 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 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Density cushion Density medium Iodixanol Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type Other;RNase A/T1 Mix RNAse concentration 0.4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 74.58 EV concentration Yes Particle yield Yes, as number of particles per million cells 2.40E+09

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EV-TRACK ID	EV200042
species	Mus musculus
sample type	Cell culture
cell type	Bone marrow-derived macrophages (BMDMs)	Immortalized bone marrow-derived macrophages (iBMDMs)
condition	Control condition	genetically modified cell line
separation protocol	DG (d)(U)C DC Filtration	DG (d)(U)C DC Filtration
Exp. nr.	1	2
EV-METRIC %	75	63