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You searched for: EV160004 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Isolation/particle analysis
Experiment number
  • Experiments differ in Isolation/particle analysis
Experiment number
  • Experiments differ in Isolation/particle analysis
Experiment number
  • Experiments differ in Isolation/particle analysis
Experiment number
  • Experiments differ in Isolation/particle analysis
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV160004 2/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 66%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
66% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Synovial fluid
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
71.08 (pelleting)
Protein markers
EV: Annexin-A1/ CD90/Thy1.1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Origin
Control condition
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
71.08
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0M
Highest density fraction
1.4M
Sample volume (mL)
1.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
60
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
138.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Annexin-A1, CD90/Thy1.1
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.
EV160004 5/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 66%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
66% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Synovial fluid
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
1421 (pelleting)
Protein markers
EV: Annexin-A1/ CD90/Thy1.1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Origin
Control condition
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
10000
Pelleting: adjusted k-factor
1421.
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0M
Highest density fraction
1.4M
Sample volume (mL)
1.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
60
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
138.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Annexin-A1, CD90/Thy1.1
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.
EV160004 1/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (30th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Synovial fluid
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
83.68 (pelleting)
Protein markers
EV: CD9/ CD44
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Origin
Control condition
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
83.68
Density gradient
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV160004 3/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (30th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Synovial fluid
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
167.3 (pelleting)
Protein markers
EV: CD9/ CD44
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Origin
Control condition
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
167.3
Density gradient
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV160004 4/5 Equus caballus Synovial fluid DG
dUC
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (30th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Synovial fluid
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
1673 (pelleting)
Protein markers
EV: CD9/ CD44
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
Origin
Control condition
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
35
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
10000
Pelleting: adjusted k-factor
1673.
Density gradient
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
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