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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200152	1/2	Homo sapiens	Serum	(d)(U)C ExoQuick	da Silva Nardi, Fabiola	2016	38%
Study summary Full title High levels of circulating extracellular vesicles with altered expression and function during pregnancy All authors Fabiola da Silva Nardi, Tatiana Ferreira Michelon, Jorge Neumann, Luis Felipe Santos Manvailer, Bettina Wagner, Peter A Horn, Maria da Graça Bicalho, Vera Rebmann Journal Immunobiology Abstract Extracellular vesicles (EVs) are widely considered important modulators of cell-cell communication a (show more...)Extracellular vesicles (EVs) are widely considered important modulators of cell-cell communication and may interact with target cells locally and on a systemic level. Several studies had shown that circulating EVs' levels are increased during pregnancy. However, EVs characteristics, composition and biological functions in pregnancy still need to be clarified. This study aims to determine if circulating EVs during pregnancy are modified regarding levels, markers and cytokine profile as well as their reactivity towards peripheral blood cells. 26 pregnant women (PW) being in the second gestational trimester and 59 non-pregnant women (NPW) were investigated. EVs enrichment was performed by ExoQuick™ or ultracentrifugation; nanoparticle tracking analysis, SDS-PAGE followed by Western Blotting and densitometry, and IFN-γ, IL-10 and TGF-β1 ELISA for EVs characterization; imaging flow cytometry to analyze EVs' uptake by peripheral blood cells and flow cytometry were performed to analyze EVs function regarding induction of caspase-3 activity. Circulating EVs' levels were increased during pregnancy [26.9×10(6)EVs/ml (range: 6.4-46.3); p=0.003] vs NPW [18.9×10(6)EVs/ml (range: 2.5-61.3)]. Importantly, the immunosuppressive TGF-β1 and IL-10 cytokine cargo were increased in EVs of PW even after normalization to 1 million EVs [TGF-β1: 0.25pg/10(6)EVs (range: 0.0-2.0); p<0.0001] and [IL-10: 0.21pg/10(6)EVs (range: 0.0-16.8); p=0.006] vs NPW. Although EVs derived from non-pregnant and pregnant women were taken up by NK cells, the latter exclusively enhanced the caspase-3 activity in CD56(dim) NK cells (8.2±0.9; p=0.02). The qualitative and quantitative pregnancy-related alterations of circulating EVs provide first hints for an immune modulating role of circulating EVs during pregnancy. (hide) EV-METRIC 38% (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 Serum 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 Commercial method Protein markers EV: CD9/ CD63/ ICAM-1/ TSG101/ HSP70/ CD81/ IFN-gamma/ IL-10/ TGF-beta 1 non-EV: CYC1 Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ ICAM-1/ TSG101/ HSP70/ CD81 Not detected contaminants CYC1 ELISA Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins IFN-gamma/ IL-10/ TGF-beta 1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 145 +/- 9.9nm EV concentration Yes

	EV200152	2/2	Homo sapiens	Serum	(d)(U)C ExoQuick	da Silva Nardi, Fabiola	2016	38%
Study summary Full title High levels of circulating extracellular vesicles with altered expression and function during pregnancy All authors Fabiola da Silva Nardi, Tatiana Ferreira Michelon, Jorge Neumann, Luis Felipe Santos Manvailer, Bettina Wagner, Peter A Horn, Maria da Graça Bicalho, Vera Rebmann Journal Immunobiology Abstract Extracellular vesicles (EVs) are widely considered important modulators of cell-cell communication a (show more...)Extracellular vesicles (EVs) are widely considered important modulators of cell-cell communication and may interact with target cells locally and on a systemic level. Several studies had shown that circulating EVs' levels are increased during pregnancy. However, EVs characteristics, composition and biological functions in pregnancy still need to be clarified. This study aims to determine if circulating EVs during pregnancy are modified regarding levels, markers and cytokine profile as well as their reactivity towards peripheral blood cells. 26 pregnant women (PW) being in the second gestational trimester and 59 non-pregnant women (NPW) were investigated. EVs enrichment was performed by ExoQuick™ or ultracentrifugation; nanoparticle tracking analysis, SDS-PAGE followed by Western Blotting and densitometry, and IFN-γ, IL-10 and TGF-β1 ELISA for EVs characterization; imaging flow cytometry to analyze EVs' uptake by peripheral blood cells and flow cytometry were performed to analyze EVs function regarding induction of caspase-3 activity. Circulating EVs' levels were increased during pregnancy [26.9×10(6)EVs/ml (range: 6.4-46.3); p=0.003] vs NPW [18.9×10(6)EVs/ml (range: 2.5-61.3)]. Importantly, the immunosuppressive TGF-β1 and IL-10 cytokine cargo were increased in EVs of PW even after normalization to 1 million EVs [TGF-β1: 0.25pg/10(6)EVs (range: 0.0-2.0); p<0.0001] and [IL-10: 0.21pg/10(6)EVs (range: 0.0-16.8); p=0.006] vs NPW. Although EVs derived from non-pregnant and pregnant women were taken up by NK cells, the latter exclusively enhanced the caspase-3 activity in CD56(dim) NK cells (8.2±0.9; p=0.02). The qualitative and quantitative pregnancy-related alterations of circulating EVs provide first hints for an immune modulating role of circulating EVs during pregnancy. (hide) EV-METRIC 38% (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 Serum Sample origin Healthy pregnant 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 Commercial method Protein markers EV: CD9/ CD63/ ICAM-1/ TSG101/ HSP70/ CD81/ IFN-gamma/ IL-10/ TGF-beta 1 non-EV: CYC1 Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ ICAM-1/ TSG101/ HSP70/ CD81 Not detected contaminants CYC1 ELISA Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins IFN-gamma/ IL-10/ TGF-beta 1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 145 +/- 9.9nm EV concentration Yes

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EV-TRACK ID	EV200152
species	Homo sapiens
sample type	Serum
condition	Control condition	Healthy pregnant
separation protocol	dUC ExoQuick	dUC ExoQuick
Exp. nr.	1	2
EV-METRIC %	38	38