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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV250037	1/2	Mus musculus	Tissue	(d)(U)C DG Filtration	Hurwitz SN	2019	67%
Study summary Full title Extraction of Extracellular Vesicles from Whole Tissue. All authors Hurwitz SN, Olcese JM, Meckes DG Journal J Vis Exp Abstract Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising t (show more...)Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins. (hide) EV-METRIC 67% (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 Tissue Sample origin Brain Focus vesicles EVs 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 Filtration Adj. k-factor 8.86 (washing) Protein markers EV: CD81/ Flotillin_2/ HSP70/ TSG101/ syntenin1/ Alix non-EV: None Proteomics yes EV density (g/ml) 1.068-1.101 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Tissue 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 MLS-50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type MLS-50 Wash: speed (g) 100000 Wash: adjusted k-factor 8.862 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10 Highest density fraction 30 Total gradient volume, incl. sample (mL) 6 Sample volume (mL) 1.5 Orientation Bottom-up Speed (g) 268000 Duration (min) 50 Fraction volume (mL) 0.49 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1.009 Filtration steps 0.45µm > x > 0.22µm, EV-subtype Distinction between multiple subtypes Density Used subtypes low density 1.057 g/ml Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins Alix/ TSG101 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 75-455 EM Image type Close-up, Wide-field

	EV250037	2/2	Mus musculus	Tissue	(d)(U)C DG Filtration	Hurwitz SN	2019	55%
Study summary Full title Extraction of Extracellular Vesicles from Whole Tissue. All authors Hurwitz SN, Olcese JM, Meckes DG Journal J Vis Exp Abstract Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising t (show more...)Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins. (hide) EV-METRIC 55% (14th 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 Tissue Sample origin Lung tumor Focus vesicles EVs 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 Filtration Adj. k-factor 8.86 (washing) Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.117-1.157 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Tissue 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 MLS-50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type MLS-50 Wash: speed (g) 100000 Wash: adjusted k-factor 8.862 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10 Highest density fraction 30 Total gradient volume, incl. sample (mL) 6 Sample volume (mL) 1.5 Orientation Bottom-up Speed (g) 268000 Duration (min) 50 Fraction volume (mL) 0.49 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1.009 Filtration steps 0.45µm > x > 0.22µm, EV-subtype Distinction between multiple subtypes Density Used subtypes high density 1.144 g/ml Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins Alix/ Flotillin-2/ HSC70/ TSG101/ syntenin1 Not detected EV-associated proteins CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV250037
species	Mus musculus
sample type	Tissue
condition	Brain	Lung tumor
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration
EV subtype	low density 1.057 g/ml	high density 1.144 g/ml
Exp. nr.	1	2
EV-METRIC %	67	55