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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170060	1/4	Bos taurus	Other	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (50th 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 Other 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 (d)(U)C Adj. k-factor 208.3 (pelleting) / 208.3 (washing) Protein markers EV: CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Other Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 1.00E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

	EV170060	2/4	Bos taurus	Serum	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (91st 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 (d)(U)C Adj. k-factor 208.3 (pelleting) / 208.3 (washing) Protein markers EV: HSP70/ CD63/ CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63, HSP70, CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 2.50E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

	EV170060	3/4	Bos taurus	Other	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (50th 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 Other 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 (d)(U)C Adj. k-factor 208.3 (pelleting) / 208.3 (washing) Protein markers EV: HSP70/ CD63/ CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Other Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63, HSP70, CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 3.00E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

	EV170060	4/4	Bos taurus	Other	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (50th 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 Other 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 (d)(U)C Adj. k-factor 208.3 (pelleting) / 208.3 (washing) Protein markers EV: HSP70/ CD63/ CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Other Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63, HSP70, CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 1.00E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

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EV-TRACK ID	EV170060
species	Bos taurus
sample type	Other	Serum	Other	Other
condition	Control condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	1	2	3	4
EV-METRIC %	55	55	55	55