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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200114	1/4	Homo sapiens	Trophoblasts	DG (d)(U)C Filtration	Elfeky O	2017	50%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 50% (87th 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 exosome 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Trophoblasts EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes

	EV200114	3/4	Homo sapiens	Blood plasma	DG (d)(U)C	Elfeky O	2017	50%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 50% (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 Blood plasma Sample origin Overweight (BMI=25-29.9kg/m2) Focus vesicles exosome 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 Density gradient (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 4.20e+7

	EV200114	4/4	Homo sapiens	Blood plasma	DG (d)(U)C	Elfeky O	2017	50%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 50% (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 Blood plasma Sample origin Obese (BMI= >30kg/m2) Focus vesicles exosome 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 Density gradient (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 6.30e+7

	EV200114	2/4	Homo sapiens	Blood plasma	DG (d)(U)C	Elfeky O	2017	44%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 44% (76th 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 Healthy pregnant Focus vesicles exosome 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 Density gradient (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 4.70e+7 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV200114
species	Homo sapiens
sample type	Cell culture	Blood plasma	Blood plasma	Blood plasma
cell type	Trophoblasts	NA	NA	NA
medium	Not specified	NA	NA	NA
condition	Control condition	Overweight (BMI=25-29.9kg/m2)	Obese (BMI= >30kg/m2)	Healthy pregnant
separation protocol	Density gradient/ dUC/ Filtration	Density gradient/ dUC/ No extra separation steps	Density gradient/ dUC/ No extra separation steps	Density gradient/ dUC/ No extra separation steps
Exp. nr.	1	3	4	2
EV-METRIC %	50	50	50	44