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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170016	1/2	Mus musculus	adipose tissue-derived macrophages	DG (d)(U)C Filtration	Ying, Wei	2017	88%
Study summary Full title Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity. All authors Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, Ofrecio JM, Wollam J, Hernandez-Carretero A, Fu W, Li P, Olefsky JM Journal Cell Abstract MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we (show more...)MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis. (hide) EV-METRIC 88% (98th 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 Obesitas Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Adj. k-factor 256 (pelleting) / 256 (washing) Protein markers EV: TSG101/ HSP70/ Syntenin1/ CD63/ CD9 non-EV: Grp94 Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells adipose tissue-derived macrophages EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS 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) 240-360 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 256.0 Wash: time (min) 20 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 256.0 Density gradient Only used for validation of main results Yes Density medium 156.9 Type Discontinuous Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 0.3 Orientation Bottom-up (sample migrates upwards) Rotor type Type 70 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 2.4 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Pelleting-wash: volume per pellet (mL) 24 Pelleting-wash: duration (min) 30 Pelleting-wash: rotor type 156.9 Pelleting-wash: speed (g) Type 70 Ti Pelleting-wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes 1.13-1.15 g/ml Used subtypes Yes Characterization: Protein analysis Protein Concentration Method DC protein assay Protein Yield (µg) 9-May Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, HSP70, TSG101, Syntenin1 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50-200 nm Extra information Full UC and density gradient protocols not in original article

	EV170016	2/2	Mus musculus	adipose tissue-derived macrophages	(d)(U)C Filtration	Ying, Wei	2017	55%
Study summary Full title Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity. All authors Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, Ofrecio JM, Wollam J, Hernandez-Carretero A, Fu W, Li P, Olefsky JM Journal Cell Abstract MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we (show more...)MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis. (hide) EV-METRIC 55% (89th 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 Filtration Adj. k-factor 256 (pelleting) / 256 (washing) Protein markers EV: TSG101/ HSP70/ Syntenin1/ CD63/ CD9 non-EV: Grp94 Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells adipose tissue-derived macrophages EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS 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) 240-360 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 256.0 Wash: time (min) 20 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 256.0 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes 1.13-1.15 g/ml Used subtypes Yes Characterization: Protein analysis Protein Concentration Method DC protein assay Protein Yield (µg) 9-May Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, HSP70, TSG101, Syntenin1 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Extra information Full UC protocol not in original article

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EV-TRACK ID	EV170016
species	Mus musculus
sample type	Cell culture
cell type	adipose tissue-derived macrophages
condition	Obesitas	Control condition
separation protocol	DG (d)(U)C Filtration	(d)(U)C Filtration
Exp. nr.	1	2
EV-METRIC %	88	55