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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190011	4/5	Mus musculus	Tissue	DG (d)(U)C	Cianciaruso C	2019	100%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 100% (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 Tissue Sample origin MC38 tumor Focus vesicles 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 DG (d)(U)C Protein markers EV: / TSG101/ CD63/ TBXAS1/ MRC1/ CD81/ COX1/ GAPDH/ CD68/ Alix/ actin-beta/ HER2/ CD9/ CD11b non-EV: Calnexin/ Gp96 Proteomics yes EV density (g/ml) 1.14 Show all info Study aim Identification of content (omics approaches)/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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 17% Highest density fraction 78% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 134000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101/ CD81/ GAPDH/ MRC1/ CD68/ actin-beta/ HER2/ TBXAS1/ COX1 Detected contaminants Calnexin/ Gp96 Flow cytometry specific beads Detected EV-associated proteins CD11b/ CD9 Flow cytometry Type of Flow cytometry Attune NxT apparatus Hardware adaptation to ~100nm EV's Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep Detected EV-associated proteins Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 190 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	5/5	Mus musculus	Tissue	DG (d)(U)C	Cianciaruso C	2019	100%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 100% (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 Tissue Sample origin E0771 tumor Focus vesicles 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 DG (d)(U)C Protein markers EV: MRC1/ COX1/ CD9/ TBXAS1 non-EV: Gp96 Proteomics yes EV density (g/ml) 1.14 Show all info Study aim Identification of content (omics approaches)/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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Density gradient Type Continuous Lowest density fraction 17% Highest density fraction 77% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 134000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ MRC1/ COX1/ TBXAS1 Detected contaminants Gp96 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	1/5	Mus musculus	MC38	(d)(U)C	Cianciaruso C	2019	78%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 78% (97th 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 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 (d)(U)C Protein markers EV: TSG101/ Syntenin1/ CD63/ CD81/ GAPDH/ Alix/ vinculin/ actin-beta/ HER2/ CD9/ CD11b non-EV: Calnexin/ Gp96 Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells MC38 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD81/ vinculin/ GAPDH/ Alix/ TSG101/ Syntenin1/ actin-beta/ CD9/ CD63 Detected contaminants Calnexin/ Gp96 Flow cytometry specific beads Detected EV-associated proteins HER2/ CD11b/ CD9 Flow cytometry Type of Flow cytometry Attune NxT apparatus Hardware adaptation to ~100nm EV's Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep Detected EV-associated proteins HER2 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 150 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	3/5	Mus musculus	Bone marrow-derived macrophages	(d)(U)C	Cianciaruso C	2019	78%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 78% (97th 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 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 (d)(U)C Protein markers EV: / TSG101/ CD63/ MRC1/ CD81/ GAPDH/ CD68/ Alix/ CD9 non-EV: Gp96 Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Bone marrow-derived macrophages EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD81/ MRC1/ GAPDH/ CD63/ TSG101/ CD9/ CD68/ MRC1/ Alix Not detected EV-associated proteins Not detected contaminants Gp96 Flow cytometry Type of Flow cytometry Attune NxT apparatus Hardware adaptation to ~100nm EV's Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep Detected EV-associated proteins Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 140 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	2/5	Mus musculus	E0771	(d)(U)C	Cianciaruso C	2019	44%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 44% (84th 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 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 (d)(U)C Protein markers EV: CD81/ Alix/ CD9/ GAPDH non-EV: Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells E0771 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ GAPDH Not detected EV-associated proteins CD81 Characterization: Lipid analysis No

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EV-TRACK ID	EV190011
species	Mus musculus
sample type	Tissue	Tissue	Cell culture	Cell culture	Cell culture
cell type	NA	NA	MC38	Bone marrow-derived macrophages	E0771
medium	NA	NA	Serum-containing but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)	Serum-containing but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)	Serum-containing but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
condition	MC38 tumor	E0771 tumor	Control condition	Control condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	4	5	1	3	2
EV-METRIC %	100	100	78	78	44