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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	Cell culture supernatant	Density gradient (Differential) (ultra)centrifugation Filtration	Omar Elfeky	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 Omar Elfeky, Sherri Longo, Andrew Lai, Gregory E Rice, Carlos Salomon 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% (81st 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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Density gradient + (Differential) (ultra)centrifugation + Filtration Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no EV density (g/ml) 1.12-1.19 Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Trophoblasts EV-harvesting Medium Not specified Separation Method Differential ultracentrifugation centrifugation steps Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 1200 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Density medium Iodixanol 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 Fluorescent NTA Relevant measurements variables specified? NA Detected EV-associated proteins PLAP/ IgG1/ CD63 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	Density gradient (Differential) (ultra)centrifugation No extra separation steps	Omar Elfeky	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 Omar Elfeky, Sherri Longo, Andrew Lai, Gregory E Rice, Carlos Salomon 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% (83rd 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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Density gradient + (Differential) (ultra)centrifugation + No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no EV density (g/ml) 1.12-1.19 Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Overweight (BMI 25-29.9kg/m2) Separation Method Differential ultracentrifugation centrifugation steps Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 1200 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Density medium Iodixanol 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 Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Particle analysis DLS Report type Mean Reported size (nm) 98 Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes

	EV200114	4/4	Homo sapiens	Blood plasma	Density gradient (Differential) (ultra)centrifugation No extra separation steps	Omar Elfeky	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 Omar Elfeky, Sherri Longo, Andrew Lai, Gregory E Rice, Carlos Salomon 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% (83rd 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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Density gradient + (Differential) (ultra)centrifugation + No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no EV density (g/ml) 1.12-1.19 Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Obese (BMI >= 30kg/m2) Separation Method Differential ultracentrifugation centrifugation steps Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 1200 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Density medium Iodixanol 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 Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Particle analysis DLS Report type Mean Reported size (nm) 105 Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes

	EV200114	2/4	Homo sapiens	Blood plasma	Density gradient (Differential) (ultra)centrifugation No extra separation steps	Omar Elfeky	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 Omar Elfeky, Sherri Longo, Andrew Lai, Gregory E Rice, Carlos Salomon 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% (77th 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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Density gradient + (Differential) (ultra)centrifugation + No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no EV density (g/ml) 1.12-1.19 Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Healthy pregnant Separation Method Differential ultracentrifugation centrifugation steps Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 1200 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Density medium Iodixanol 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 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Fluorescent NTA Relevant measurements variables specified? NA Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Particle analysis DLS Report type Mean Reported size (nm) 102 Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up

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