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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230697	1/3	Porphyromonas gingivalis	W50	(d)(U)C DG Filtration UF	Cecil JD	2016	67%
Study summary Full title Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens. All authors Cecil JD, O'Brien-Simpson NM, Lenzo JC, Holden JA, Chen YY, Singleton W, Gause KT, Yan Y, Caruso F, Reynolds EC Journal PLoS One Abstract Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivali (show more...)Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. (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 Control condition Focus vesicles outer membrane vesicles 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Porphyromonas gingivalis Sample Type Cell culture supernatant EV-producing cells W50 EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type JA-30.50 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 15% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) not spec Orientation Bottom-up Speed (g) 150000 Duration (min) 960 Fraction volume (mL) 1.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 150000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Characterization: RNA analysis RNA analysis Type Qubit RNA assay kit Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 121 Particle analysis: flow cytometry Flow cytometer type Apogee A50-Micro Flow Cytometer Hardware adjustment Calibration bead size 0.11 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.98E+12 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 50-150

	EV230697	2/3	Treponema denticola	ATCC 35405	(d)(U)C DG Filtration UF	Cecil JD	2016	67%
Study summary Full title Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens. All authors Cecil JD, O'Brien-Simpson NM, Lenzo JC, Holden JA, Chen YY, Singleton W, Gause KT, Yan Y, Caruso F, Reynolds EC Journal PLoS One Abstract Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivali (show more...)Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. (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 Control condition Focus vesicles outer membrane vesicles 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Treponema denticola Sample Type Cell culture supernatant EV-producing cells ATCC 35405 EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type JA-30.50 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 15% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) not spec Orientation Bottom-up Speed (g) 150000 Duration (min) 2880 Fraction volume (mL) 1.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 150000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Characterization: RNA analysis RNA analysis Type Qubit RNA assay kit Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 75 Particle analysis: flow cytometry Flow cytometer type Apogee A50-Micro Flow Cytometer Hardware adjustment Calibration bead size 0.11 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.54E+11 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 30-120

	EV230697	3/3	Tannerella forsythia	ATCC 43037	(d)(U)C DG Filtration UF	Cecil JD	2016	67%
Study summary Full title Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens. All authors Cecil JD, O'Brien-Simpson NM, Lenzo JC, Holden JA, Chen YY, Singleton W, Gause KT, Yan Y, Caruso F, Reynolds EC Journal PLoS One Abstract Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivali (show more...)Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. (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 Control condition Focus vesicles outer membrane vesicles 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Tannerella forsythia Sample Type Cell culture supernatant EV-producing cells ATCC 43037 EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type JA-30.50 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 15% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) not spec Orientation Bottom-up Speed (g) 150000 Duration (min) 2880 Fraction volume (mL) 1.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 150000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Characterization: RNA analysis RNA analysis Type Qubit RNA assay kit Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 158 Particle analysis: flow cytometry Flow cytometer type Apogee A50-Micro Flow Cytometer Hardware adjustment Calibration bead size 0.11 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.00E+12 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 80-250

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EV-TRACK ID	EV230697
species	Porphyromonas gingivalis	Treponema denticola	Tannerella forsythia
sample type	Cell culture	Cell culture	Cell culture
cell type	W50	ATCC 35405	ATCC 43037
condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration
Exp. nr.	1	2	3
EV-METRIC %	67	67	67