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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200073	2/4	Homo sapiens	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC DG	Bordas, Marie	2020	88%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 88% (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 Solid Tissue Sample origin CLL 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 Size-exclusion chromatopraphy (IZON) DC DG Protein markers EV: TSG101/ CD81/ Flotillin1/ CD9 non-EV: cytochrome C/ GM130/ Calreticulin Proteomics no EV density (g/ml) 1.12 Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 0% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 7 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100 Duration (min) 120 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) 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 Flotillin1/ CD9/ TSG101/ CD81 Detected contaminants Calreticulin Not detected contaminants cytochrome C/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.00E+10 EM EM-type Immuno-EM/ Transmission-EM EM protein Other;HLA-DR Image type Close-up

	EV200073	4/4	Mus musculus	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC DG	Bordas, Marie	2020	88%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 88% (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 Solid Tissue Sample origin CLL 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 Size-exclusion chromatopraphy (IZON) DC DG Protein markers EV: Alix/ TSG101/ Flotillin1 non-EV: ATPA5/ Calreticulin Proteomics no EV density (g/ml) 1.12 Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 0% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 7 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100 Duration (min) 120 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) 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 Flotillin1/ Alix/ TSG101 Detected contaminants Calreticulin Not detected contaminants ATPA5 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 7.00E+09 EM EM-type Transmission-EM Image type Close-up

	EV200073	1/4	Homo sapiens	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON)	Bordas, Marie	2020	63%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 63% (25th 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 Solid Tissue Sample origin CLL 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 Size-exclusion chromatopraphy (IZON) DC SEC (IZON) Protein markers EV: TSG101/ CD81/ Flotillin1/ CD9 non-EV: Cytochrome C/ GM130/ Calreticulin Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) Other Name other separation method SEC (IZON) 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 Flotillin1/ CD9/ TSG101/ CD81 Detected contaminants Calreticulin Not detected contaminants Cytochrome C/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.00E+10 EM EM-type Immuno-EM/ Transmission-EM EM protein Other;HLA-DR Image type Close-up

	EV200073	3/4	Mus musculus	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON)	Bordas, Marie	2020	63%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 63% (25th 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 Solid Tissue Sample origin CLL 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 Size-exclusion chromatopraphy (IZON) DC SEC (IZON) Protein markers EV: Alix/ TSG101/ CD81 non-EV: ATPA5/ Calreticulin Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) Other Name other separation method SEC (IZON) 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 Alix/ TSG101/ CD81 Detected contaminants Calreticulin Not detected contaminants ATPA5 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.50E+10 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV200073
species	Homo sapiens	Mus musculus	Homo sapiens	Mus musculus
sample type	Solid Tissue	Solid Tissue	Solid Tissue	Solid Tissue
condition	CLL	CLL	CLL	CLL
separation protocol	(d)(U)C Size-exclusion chromatopraphy (IZON) DC DG	(d)(U)C Size-exclusion chromatopraphy (IZON) DC DG	(d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON)	(d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON)
Exp. nr.	2	4	1	3
EV-METRIC %	88	88	63	63