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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220363	4/4	Rattus norvegicus	PC12	(d)(U)C DG	Tedford E	2023	71%
Study summary Full title Infection-induced extracellular vesicles evoke neuronal transcriptional and epigenetic changes. All authors Tedford E, Badya NB, Laing C, Asaoka N, Kaneko S, Filippi BM, McConkey GA Journal Sci Rep Abstract Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammati (show more...)Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function. (hide) EV-METRIC 71% (96th 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 Toxoplasma gondii Prugniaud-infected 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 Density gradient Protein markers EV: CD63/ CD81/ Flotillin-1/ TSG101/ EpCAM/ Alix non-EV: GM130 Proteomics no EV density (g/ml) 1.16 Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells PC12 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 160000 Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 10% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 70000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 160000 Pelleting: adjusted k-factor 1.132 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Other 1 Dot Blot SBI Detected EV-associated proteins CD63/ CD81/ Flotillin1/ TSG101/ EpCAM Not detected EV-associated proteins Alix Detected contaminants none Not detected contaminants GM130 Characterization: RNA analysis RNA analysis Type RT-PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 100

	EV220363	1/4	Homo sapiens	BE(2)-M17	(d)(U)C DG	Tedford E	2023	43%
Study summary Full title Infection-induced extracellular vesicles evoke neuronal transcriptional and epigenetic changes. All authors Tedford E, Badya NB, Laing C, Asaoka N, Kaneko S, Filippi BM, McConkey GA Journal Sci Rep Abstract Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammati (show more...)Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function. (hide) EV-METRIC 43% (82nd 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 Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.16 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells BE(2)-M17 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 160000 Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 10% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 70000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 160000 Pelleting: adjusted k-factor 1.132 Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220363	2/4	Homo sapiens	Human foreskin fibroblasts	(d)(U)C DG	Tedford E	2023	43%
Study summary Full title Infection-induced extracellular vesicles evoke neuronal transcriptional and epigenetic changes. All authors Tedford E, Badya NB, Laing C, Asaoka N, Kaneko S, Filippi BM, McConkey GA Journal Sci Rep Abstract Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammati (show more...)Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function. (hide) EV-METRIC 43% (82nd 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 Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.16 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 160000 Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 10% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 70000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 160000 Pelleting: adjusted k-factor 1.132 Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220363	3/4	Rattus norvegicus	PC12	(d)(U)C DG	Tedford E	2023	43%
Study summary Full title Infection-induced extracellular vesicles evoke neuronal transcriptional and epigenetic changes. All authors Tedford E, Badya NB, Laing C, Asaoka N, Kaneko S, Filippi BM, McConkey GA Journal Sci Rep Abstract Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammati (show more...)Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function. (hide) EV-METRIC 43% (82nd 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 Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.16 Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells PC12 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 160000 Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 10% Highest density fraction 70% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 70000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 160000 Pelleting: adjusted k-factor 1.132 Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Characterization: RNA analysis RNA analysis Type RT-PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 100

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EV-TRACK ID	EV220363
species	Rattus norvegicus	Homo sapiens	Homo sapiens	Rattus norvegicus
sample type	Cell culture	Cell culture	Cell culture	Cell culture
cell type	PC12	BE(2)-M17	Human foreskin fibroblasts	PC12
condition	Toxoplasma gondii Prugniaud-infected	Control condition	Control condition	Control condition
separation protocol	dUC/ Density gradient	dUC/ Density gradient	dUC/ Density gradient	dUC/ Density gradient
Exp. nr.	4	1	2	3
EV-METRIC %	71	43	43	43