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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200099	1/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin raw 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	2/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin raw 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	3/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin pasteurized 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	4/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin pasteurized 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	5/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin pasteurized and homogenized 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	6/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin pasteurized and homogenized 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	7/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin UHT 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.20-1.24 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.20-1.24 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

	EV200099	8/8	Bos taurus	milk	(d)(U)C DG	Kleinjan, Marije	2021	100%
Study summary Full title Regular Industrial Processing of Bovine Milk Impacts the Integrity and Molecular Composition of Extracellular Vesicles All authors Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben Journal J Nutr Abstract Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide) EV-METRIC 100% (93rd 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 milk Sample origin UHT 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 (d)(U)C DG Protein markers EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9 non-EV: beta-lactoglobulin/ beta-casein Proteomics no EV density (g/ml) 1.18-1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Density Used subtypes 1.18-1.13 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101 Not detected contaminants beta-casein/ beta-lactoglobulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) Hardware adjustment High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles Calibration bead size 0.1 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 200

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EV-TRACK ID	EV200099
species	Bos taurus
sample type	milk
condition	raw	raw	pasteurized	pasteurized	pasteurized and homogenized	pasteurized and homogenized	UHT	UHT
separation protocol	(d)(U)C DG	(d)(U)C DG	(d)(U)C DG	(d)(U)C DG	(d)(U)C DG	(d)(U)C DG	(d)(U)C DG	(d)(U)C DG
EV subtype	1.20-1.24	1.18-1.13	1.20-1.24	1.18-1.13	1.20-1.24	1.18-1.13	1.20-1.24	1.18-1.13
Exp. nr.	1	2	3	4	5	6	7	8
EV-METRIC %	100	100	100	100	100	100	100	100

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