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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220036	2/3	Canis familiaris	CYPp	(d)(U)C	Sammarco A	2018	44%
Study summary Full title Preliminary investigation of extracellular vesicles in mammary cancer of dogs and cats: Identification and characterization. All authors Sammarco A, Finesso G, Cavicchioli L, Ferro S, Caicci F, Zanetti R, Sacchetto R, Zappulli V Journal Vet Comp Oncol Abstract Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role (show more...)Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role in cell-to-cell communication. They exert pleiotropic biological functions via the horizontal transfer of bioactive molecules (DNA, RNAs, proteins, and lipids) within the tumour microenvironment and throughout the body. In human cancer, EVs are known to interfere with pathways that lead to tumour progression and are used as novel cancer biomarkers. In veterinary medicine, very little is known on cancer-derived EVs. In this study, we preliminarily characterized EVs in mammary gland cancer of dogs and cats. EVs were isolated by ultracentrifugation from canine (CYPp), feline (FMCp) and human (MCF7) mammary tumour cell lines. EVs were visualized by transmission electron microscopy (TEM), counted using nanoparticle tracking analysis (NTA) and characterized by immunogold (CD63 and Alix) and western blot (Alix and TSG101). Additionally, EV production by "donor" cells (palmtdTomato ) and uptake by "recipient" cells (GFP ) were assessed. EVs were successfully isolated from all 3 cell lines by ultracentrifugation. Membrane-bound structures (50-400 nm) were identified by TEM and were positive for both CD63 and Alix at immunogold. Western blot showed positivity of EVs to Alix and TSG101. NTA analysis detected EVs from cell culture media ranging from 1.67 to 2.56 × 10 as number of EVs/cell and from 80 to 600 nm in size. Confocal microscopy identified the presence of palmtdTomato EVs into the cytoplasm of GFP cells. This preliminary study identified and characterized canine and feline mammary tumour cell-derived EVs, opening in veterinary medicine a new interesting unexplored field with several applications and limitless potential. (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 (Differential) (ultra)centrifugation Protein markers EV: TSG101/ Alix non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Canis familiaris Sample Type Cell culture supernatant EV-producing cells CYPp EV-harvesting Medium EV-depleted medium Preparation of EDS 16h at 100,000g + 0.22 um filtration/ Other preparation Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 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 TSG101/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per million cells: 2.56e+8 EM EM-type Immuno-EM/ Transmission-EM EM protein Other/ CD63/ Alix Image type Close-up, Wide-field

	EV220036	1/3	Homo sapiens	MCF7	(d)(U)C	Sammarco A	2018	22%
Study summary Full title Preliminary investigation of extracellular vesicles in mammary cancer of dogs and cats: Identification and characterization. All authors Sammarco A, Finesso G, Cavicchioli L, Ferro S, Caicci F, Zanetti R, Sacchetto R, Zappulli V Journal Vet Comp Oncol Abstract Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role (show more...)Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role in cell-to-cell communication. They exert pleiotropic biological functions via the horizontal transfer of bioactive molecules (DNA, RNAs, proteins, and lipids) within the tumour microenvironment and throughout the body. In human cancer, EVs are known to interfere with pathways that lead to tumour progression and are used as novel cancer biomarkers. In veterinary medicine, very little is known on cancer-derived EVs. In this study, we preliminarily characterized EVs in mammary gland cancer of dogs and cats. EVs were isolated by ultracentrifugation from canine (CYPp), feline (FMCp) and human (MCF7) mammary tumour cell lines. EVs were visualized by transmission electron microscopy (TEM), counted using nanoparticle tracking analysis (NTA) and characterized by immunogold (CD63 and Alix) and western blot (Alix and TSG101). Additionally, EV production by "donor" cells (palmtdTomato ) and uptake by "recipient" cells (GFP ) were assessed. EVs were successfully isolated from all 3 cell lines by ultracentrifugation. Membrane-bound structures (50-400 nm) were identified by TEM and were positive for both CD63 and Alix at immunogold. Western blot showed positivity of EVs to Alix and TSG101. NTA analysis detected EVs from cell culture media ranging from 1.67 to 2.56 × 10 as number of EVs/cell and from 80 to 600 nm in size. Confocal microscopy identified the presence of palmtdTomato EVs into the cytoplasm of GFP cells. This preliminary study identified and characterized canine and feline mammary tumour cell-derived EVs, opening in veterinary medicine a new interesting unexplored field with several applications and limitless potential. (hide) EV-METRIC 22% (59th 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 (Differential) (ultra)centrifugation Protein markers EV: TSG101/ Alix non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Preparation of EDS 16h at 100,000g + 0.22 um filtration/ Other preparation Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 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 TSG101/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per million cells: 2.52e+8

	EV220036	3/3	Felis silvestris catus	FMCp	(d)(U)C	Sammarco A	2018	22%
Study summary Full title Preliminary investigation of extracellular vesicles in mammary cancer of dogs and cats: Identification and characterization. All authors Sammarco A, Finesso G, Cavicchioli L, Ferro S, Caicci F, Zanetti R, Sacchetto R, Zappulli V Journal Vet Comp Oncol Abstract Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role (show more...)Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role in cell-to-cell communication. They exert pleiotropic biological functions via the horizontal transfer of bioactive molecules (DNA, RNAs, proteins, and lipids) within the tumour microenvironment and throughout the body. In human cancer, EVs are known to interfere with pathways that lead to tumour progression and are used as novel cancer biomarkers. In veterinary medicine, very little is known on cancer-derived EVs. In this study, we preliminarily characterized EVs in mammary gland cancer of dogs and cats. EVs were isolated by ultracentrifugation from canine (CYPp), feline (FMCp) and human (MCF7) mammary tumour cell lines. EVs were visualized by transmission electron microscopy (TEM), counted using nanoparticle tracking analysis (NTA) and characterized by immunogold (CD63 and Alix) and western blot (Alix and TSG101). Additionally, EV production by "donor" cells (palmtdTomato ) and uptake by "recipient" cells (GFP ) were assessed. EVs were successfully isolated from all 3 cell lines by ultracentrifugation. Membrane-bound structures (50-400 nm) were identified by TEM and were positive for both CD63 and Alix at immunogold. Western blot showed positivity of EVs to Alix and TSG101. NTA analysis detected EVs from cell culture media ranging from 1.67 to 2.56 × 10 as number of EVs/cell and from 80 to 600 nm in size. Confocal microscopy identified the presence of palmtdTomato EVs into the cytoplasm of GFP cells. This preliminary study identified and characterized canine and feline mammary tumour cell-derived EVs, opening in veterinary medicine a new interesting unexplored field with several applications and limitless potential. (hide) EV-METRIC 22% (59th 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ TSG101 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Felis silvestris catus Sample Type Cell culture supernatant EV-producing cells FMCp EV-harvesting Medium EV-depleted medium Preparation of EDS 16h at 100,000g + 0.22 um filtration/ Other preparation Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 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 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per million cells: 1.67e+8

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EV-TRACK ID	EV220036
species	Canis familiaris	Homo sapiens	Felis silvestris catus
sample type	Cell culture	Cell culture	Cell culture
cell type	CYPp	MCF7	FMCp
condition	Control condition	Control condition	Control condition
separation protocol	dUC	dUC	dUC
Exp. nr.	2	1	3
EV-METRIC %	44	22	22