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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220318	1/4	Mus musculus	CAD5	(d)(U)C	Jakub Soukup	2023	67%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs. (hide) EV-METRIC 67% (94th 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 Prion infected cell culture 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 Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ CD81/ PrP non-EV: Calnexin Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells CAD5 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g 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 Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 19827 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 19556.1 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ PrP Not detected EV-associated proteins CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Extra information We have isolated and analysed Large and Small extracellular vesicles from the same conditioned medium aiming to separate them and use them for infection. Large EVs supposed to be above 70 nm in size and enriched in Beta 1 integrin while Small EVs are 30-150 nm and depleted in Beta 1 integrin. Secondary cell culture served as control.

	EV220318	2/4	Mus musculus	CAD5	(d)(U)C DC	Jakub Soukup	2023	67%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs. (hide) EV-METRIC 67% (94th 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 Prion infected cell culture 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 Density cushion Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ CD81/ PrP non-EV: Calnexin/ Integrin-beta1 Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells CAD5 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g 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 SW 28 Pelleting: speed (g) 112398.1 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 110862.3 Density cushion Density medium Sucrose Sample volume 11 Cushion volume 2.5 Density of the cushion 40% Centrifugation time 70 Centrifugation speed 110862.3 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ PrP Not detected EV-associated proteins CD81 Detected contaminants Integrin-beta1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 79

	EV220318	3/4	Mus musculus	PK1	(d)(U)C	Jakub Soukup	2023	56%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs. (hide) EV-METRIC 56% (90th 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 Prion infected cell culture 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 Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ CD81/ PrP non-EV: Calnexin Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells PK1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g 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 Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 19827 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 19556.1 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ PrP Not detected EV-associated proteins CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV220318	4/4	Mus musculus	PK1	(d)(U)C DC	Jakub Soukup	2023	56%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs. (hide) EV-METRIC 56% (90th 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 Prion infected cell culture 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 Density cushion Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ CD81/ PrP non-EV: Calnexin/ Integrin-beta1 Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells PK1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g 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 SW 28 Pelleting: speed (g) 112398.1 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 110862.3 Density cushion Density medium Sucrose Sample volume 11 Cushion volume 2.5 Density of the cushion 40% Centrifugation time 70 Centrifugation speed 110862.3 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ PrP Not detected EV-associated proteins CD81 Detected contaminants Integrin-beta1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV220318
species	Mus musculus
sample type	Cell culture
cell type	CAD5	CAD5	PK1	PK1
condition	Prion infected cell culture	Prion infected cell culture	Prion infected cell culture	Prion infected cell culture
separation protocol	dUC	dUC/ DC	dUC	dUC/ DC
vesicle related term	large EV	small EV	large EV	small EV
Exp. nr.	1	2	3	4
EV-METRIC %	67	67	56	56