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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210043	1/2	Homo sapiens	hcMEC/D3	DG (d)(U)C UF Filtration	Fikatas, Antonios	2021	100%
Study summary Full title Deciphering the Role of Extracellular Vesicles Derived from ZIKV-Infected hcMEC/D3 Cells on the Blood-Brain Barrier System All authors Antonios Fikatas, Jonas Dehairs, Sam Noppen, Jordi Doijen, Frank Vanderhoydonc, Eef Meyen, Johannes V Swinnen, Christophe Pannecouque, Dominique Schols Journal Viruses Abstract To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechani (show more...)To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechanisms underlying ZIKV-associated pathogenesis of the central nervous system (CNS) are largely unexplored. Getting more insight into the cellular pathways that ZIKV recruits to facilitate infection of susceptible cells will be crucial for establishing an effective treatment strategy. In general, cells secrete a number of vesicles, known as extracellular vesicles (EVs), in response to viral infections. These EVs serve as intercellular communicators. Here, we investigated the role of EVs derived from ZIKV-infected human brain microvascular endothelial cells on the blood–brain barrier (BBB) system. We demonstrated that ZIKV-infected EVs (IEVs) can incorporate viral components, including ZIKV RNA, NS1, and E-protein, and further transfer them to several types of CNS cells. Using label-free impedance-based biosensing, we observed that ZIKV and IEVs can temporally disturb the monolayer integrity of BBB-mimicking cells, possibly by inducing structural rearrangements of the adherent protein VE-cadherin (immunofluorescence staining). Finally, differences in the lipidomic profile between EVs and their parental cells possibly suggest a preferential sorting mechanism of specific lipid species into the vesicles. To conclude, these data suggest that IEVs could be postulated as vehicles (Trojan horse) for ZIKV transmission via the BBB. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C UF Filtration Protein markers EV: TSG101/ Alix/ CD63 non-EV: Calnexin Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hcMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 1260 Pelleting: rotor type TH-641 Pelleting: speed (g) 100 000 Wash: volume per pellet (ml) 10 Wash: time (min) 180 Wash: Rotor Type TH-641 Wash: speed (g) 100 000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type TH-641 Speed (g) 100 000 Duration (min) 1080 Fraction volume (mL) 1,5 Fraction processing None Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 165 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1,50E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 130

	EV210043	2/2	Homo sapiens	hcMEC/D3	DG (d)(U)C UF Filtration	Fikatas, Antonios	2021	89%
Study summary Full title Deciphering the Role of Extracellular Vesicles Derived from ZIKV-Infected hcMEC/D3 Cells on the Blood-Brain Barrier System All authors Antonios Fikatas, Jonas Dehairs, Sam Noppen, Jordi Doijen, Frank Vanderhoydonc, Eef Meyen, Johannes V Swinnen, Christophe Pannecouque, Dominique Schols Journal Viruses Abstract To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechani (show more...)To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechanisms underlying ZIKV-associated pathogenesis of the central nervous system (CNS) are largely unexplored. Getting more insight into the cellular pathways that ZIKV recruits to facilitate infection of susceptible cells will be crucial for establishing an effective treatment strategy. In general, cells secrete a number of vesicles, known as extracellular vesicles (EVs), in response to viral infections. These EVs serve as intercellular communicators. Here, we investigated the role of EVs derived from ZIKV-infected human brain microvascular endothelial cells on the blood–brain barrier (BBB) system. We demonstrated that ZIKV-infected EVs (IEVs) can incorporate viral components, including ZIKV RNA, NS1, and E-protein, and further transfer them to several types of CNS cells. Using label-free impedance-based biosensing, we observed that ZIKV and IEVs can temporally disturb the monolayer integrity of BBB-mimicking cells, possibly by inducing structural rearrangements of the adherent protein VE-cadherin (immunofluorescence staining). Finally, differences in the lipidomic profile between EVs and their parental cells possibly suggest a preferential sorting mechanism of specific lipid species into the vesicles. To conclude, these data suggest that IEVs could be postulated as vehicles (Trojan horse) for ZIKV transmission via the BBB. (hide) EV-METRIC 89% (99th 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 ZIKV-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 DG (d)(U)C UF Filtration Protein markers EV: TSG101/ Alix/ CD63 non-EV: Calnexin Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hcMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 1260 Pelleting: rotor type TH-641 Pelleting: speed (g) 100 000 Wash: volume per pellet (ml) 10 Wash: time (min) 180 Wash: Rotor Type TH-641 Wash: speed (g) 100 000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Top-down Rotor type TH-641 Speed (g) 100 000 Duration (min) 1080 Fraction volume (mL) 1,5 Fraction processing None Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1,40E+08 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 130

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EV-TRACK ID	EV210043
species	Homo sapiens
sample type	Cell culture
cell type	hcMEC/D3
condition	Control condition	ZIKV-infected
separation protocol	DG (d)(U)C UF Filtration	DG (d)(U)C UF Filtration
Exp. nr.	1	2
EV-METRIC %	100	89