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You searched for: EV230994 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
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
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV230994 2/6 Bos taurus Commercially available FBS (d)(U)C Urzì O 2024 67%

Study summary

Full title
All authors
Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R
Journal
J Extracell Vesicles
Abstract
The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide)
EV-METRIC
67% (75th 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
Commercially available FBS
Sample origin
HI-before EV-depl
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: HSP70/ HSP90/ HBA1
non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Commercially available FBS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
HSP70/ HSP90/ HBA1
Proteomics database
No
Detected contaminants
Albumin/ Argonaute-2/ Calreticulin
Not detected contaminants
GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 32260000
EM
EM-type
Transmission-EM
Image type
Close-up
EV230994 4/6 Bos taurus Commercially available FBS (d)(U)C Urzì O 2024 67%

Study summary

Full title
All authors
Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R
Journal
J Extracell Vesicles
Abstract
The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide)
EV-METRIC
67% (75th 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
Commercially available FBS
Sample origin
no-HI
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: HSP70/ HSP90/ HBA1
non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Commercially available FBS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
HSP70/ HSP90/ HBA1
Proteomics database
No
Detected contaminants
Albumin/ Argonaute-2/ Calreticulin
Not detected contaminants
GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 148220000
EM
EM-type
Transmission-EM
Image type
Close-up
EV230994 6/6 Bos taurus Commercially available FBS (d)(U)C Urzì O 2024 67%

Study summary

Full title
All authors
Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R
Journal
J Extracell Vesicles
Abstract
The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide)
EV-METRIC
67% (75th 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
Commercially available FBS
Sample origin
HI-after EV-depl
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: HSP70/ HSP90/ HBA1
non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Commercially available FBS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
HSP70/ HSP90/ HBA1
Proteomics database
No
Detected contaminants
Albumin/ Argonaute-2/ Calreticulin
Not detected contaminants
GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 133940000
EM
EM-type
Transmission-EM
Image type
Close-up
EV230994 1/6 Bos taurus Commercially available FBS (d)(U)C Urzì O 2024 29%

Study summary

Full title
All authors
Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R
Journal
J Extracell Vesicles
Abstract
The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide)
EV-METRIC
29% (25th 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
Commercially available FBS
Sample origin
no-HI
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Commercially available FBS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 74680000
EM
EM-type
Transmission-EM
Image type
Close-up
EV230994 3/6 Bos taurus Commercially available FBS (d)(U)C Urzì O 2024 29%

Study summary

Full title
All authors
Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R
Journal
J Extracell Vesicles
Abstract
The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide)
EV-METRIC
29% (25th 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
Commercially available FBS
Sample origin
HI-after EV-depl
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Commercially available FBS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 21600000
EM
EM-type
Transmission-EM
Image type
Close-up
EV230994 5/6 Bos taurus Commercially available FBS (d)(U)C Urzì O 2024 29%

Study summary

Full title
All authors
Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R
Journal
J Extracell Vesicles
Abstract
The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide)
EV-METRIC
29% (25th 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
Commercially available FBS
Sample origin
HI-before EV-depl
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Commercially available FBS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
118000
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 24400000
EM
EM-type
Transmission-EM
Image type
Close-up
1 - 6 of 6
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV230994
species
Bos
taurus
sample type
Commercially
available
FBS
condition
HI-before
EV-depl
no-HI
HI-after
EV-depl
no-HI
HI-after
EV-depl
HI-before
EV-depl
separation protocol
dUC
dUC
dUC
dUC
dUC
dUC
Exp. nr.
2
4
6
1
3
5
EV-METRIC %
67
67
67
29
29
29