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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV240137	2/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	67%
Study summary Full title Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway All authors Luis A. Arteaga-Blanco, Andrew E. Evans, Dan A. Dixon Journal Cells Abstract NA (show more...)NA (hide) EV-METRIC 67% (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 Blood plasma Sample origin Control condition Focus vesicles small 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: CD63/ CD81/ actin-beta non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus 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: rotor type S55-S Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 2 Wash: time (min) 70 Wash: Rotor Type S55-S Wash: speed (g) 150000 Filtration steps 0.8 µm/ 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD63/ CD81/ actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.10E+08 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 120

	EV240137	4/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	67%
Study summary Full title Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway All authors Luis A. Arteaga-Blanco, Andrew E. Evans, Dan A. Dixon Journal Cells Abstract NA (show more...)NA (hide) EV-METRIC 67% (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 Blood plasma Sample origin APCMin/+ CRC mice model Focus vesicles small 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: CD63/ CD81/ actin-beta non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus 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: rotor type S55-S Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 2 Wash: time (min) 70 Wash: Rotor Type S55-S Wash: speed (g) 150000 Filtration steps 0.8 µm/ 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD63/ CD81/ actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.36E+09 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 132

	EV240137	1/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	56%
Study summary Full title Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway All authors Luis A. Arteaga-Blanco, Andrew E. Evans, Dan A. Dixon Journal Cells Abstract NA (show more...)NA (hide) EV-METRIC 56% (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 Blood plasma Sample origin Control condition Focus vesicles large 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: CD63/ CD81/ actin-beta non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus 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 Pelleting performed Yes Pelleting: speed (g) 15000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 40 Wash: speed (g) 15000 Filtration steps 0.8 µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD63/ CD81/ actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 191 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.29E+08 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 246 Extra information This study characterizes plasma-derived EV subtypes (large and small EVs) and other non-vesicular entities released from the colorectal (CRC) mouse model APCMin/+. EV preparations, including large EVs (LEV) and small EVs (SEV), were characterized based on their physical (NTA and SEM) and biochemical properties (WB). Because we did not use additional purification methodologies such as size exclusion chromatography, density gradient, immunocapture, etc., we expected our crude EV preparations to be contaminated with other extracellular particles (e.g., lipoproteins) and soluble proteins such as albumin, immunoglobulins, and fibrinogen. Therefore, the operational terms EV preparation or EV-containing preparations were used to refer to nanoparticles obtained by multiple centrifugation steps containing populations of LEV or SEV and non-vesicular entities. We believe this is an excellent opportunity to share our protocol for reproducibility purposes with other researchers working with plasma-

	EV240137	3/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	56%
Study summary Full title Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway All authors Luis A. Arteaga-Blanco, Andrew E. Evans, Dan A. Dixon Journal Cells Abstract NA (show more...)NA (hide) EV-METRIC 56% (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 Blood plasma Sample origin APCMin/+ CRC mice model Focus vesicles large 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: CD63/ CD81/ actin-beta non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus 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 Pelleting performed Yes Pelleting: speed (g) 15000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 40 Wash: speed (g) 15000 Filtration steps 0.8 µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD63/ CD81/ actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 246 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.41E+09 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 290

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EV-TRACK ID	EV240137
species	Mus musculus
sample type	Blood plasma
condition	Control condition	APCMin/+ CRC mice model	Control condition	APCMin/+ CRC mice model
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
vesicle related term	small EV	small EV	large EV	large EV
Exp. nr.	2	4	1	3
EV-METRIC %	67	67	56	56