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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110077	3/3	Homo sapiens	Blood plasma	(d)(U)C Filtration	Grant R	2011	33%
Study summary Full title A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma All authors Grant R, Ansa-Addo E, Stratton D, Antwi-Baffour S, Jorfi S, Kholia S, Krige L, Lange S, Inal J Journal J Immunol Methods Abstract The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably (show more...)The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 ?m in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 ?m pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 ?m) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 ?M) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects. (hide) EV-METRIC 33% (66th 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 Blood plasma Sample origin NAY Focus vesicles exosomes / "Membrane(-derived) 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 (d)(U)C Filtration Protein markers EV: CD63/ Annexin5 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.2µm > x > 0.1µm Western Blot Antibody details provided? No Detected EV-associated proteins Annexin5 ELISA Antibody details provided? No Detected EV-associated proteins Annexin5 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV110077	1/3	Homo sapiens	Blood plasma	(d)(U)C	Grant R	2011	0%
Study summary Full title A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma All authors Grant R, Ansa-Addo E, Stratton D, Antwi-Baffour S, Jorfi S, Kholia S, Krige L, Lange S, Inal J Journal J Immunol Methods Abstract The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably (show more...)The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 ?m in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 ?m pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 ?m) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 ?M) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects. (hide) EV-METRIC 0% (median: 22% 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 Blood plasma Sample origin NAY Focus vesicles exosomes / "Membrane(-derived) 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 (d)(U)C Protein markers EV: CD63/ Annexin5 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma 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: time(min) 60 Western Blot Antibody details provided? No Detected EV-associated proteins Annexin5 ELISA Antibody details provided? No Detected EV-associated proteins Annexin5 Characterization: Particle analysis

	EV110077	2/3	Homo sapiens	Blood plasma	(d)(U)C	Grant R	2011	0%
Study summary Full title A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma All authors Grant R, Ansa-Addo E, Stratton D, Antwi-Baffour S, Jorfi S, Kholia S, Krige L, Lange S, Inal J Journal J Immunol Methods Abstract The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably (show more...)The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 ?m in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 ?m pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 ?m) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 ?M) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects. (hide) EV-METRIC 0% (median: 22% 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 Blood plasma Sample origin NAY Focus vesicles exosomes / "Membrane(-derived) 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 (d)(U)C Protein markers EV: CD63/ Annexin5 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Western Blot Antibody details provided? No Detected EV-associated proteins Annexin5 ELISA Antibody details provided? No Detected EV-associated proteins Annexin5 Characterization: Particle analysis

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EV-TRACK ID	EV110077
species	Homo sapiens
sample type	Blood plasma
condition	NAY
separation protocol	(d)(U)C Filtration	(d)(U)C	(d)(U)C
Exp. nr.	3	1	2
EV-METRIC %	33	0	0