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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
EV230597 1/9 Homo sapiens ESC (d)(U)C
DG
Chen Z 2024 100%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
100% (99th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Density gradient
Adj. k-factor
37432 (washing)
Protein markers
EV: Alix/ CD9/ CD63/ TSG101/ CD81
non-EV: GM130/ Calnexin
Proteomics
yes
EV density (g/ml)
1.17
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
ESC
EV-harvesting Medium
Serum free medium
Cell count
2.00E+07
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
25
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
37432
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
20%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
11
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
150000
Duration (min)
600
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: speed (g)
150000
Pelleting: adjusted k-factor
10018
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected contaminants
GM130/ Calnexin
Flow cytometry
Type of Flow cytometry
Nano-flow Cytometry
Hardware adaptation to ~100nm EV's
By utilizing Rayleigh scattering
Calibration bead size
0.25
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Proteomics database
ProteomeXchange
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Nano-flow Cytometry
Hardware adjustment
By utilizing Rayleigh scattering
Calibration bead size
68/ 91,113,155
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.03E+08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100
EV230597 2/9 Homo sapiens iPSC (d)(U)C
DG
Chen Z 2024 100%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
100% (99th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Density gradient
Adj. k-factor
37432 (washing)
Protein markers
EV: TSG101/ CD9/ CD63/ CD81
non-EV: None
Proteomics
yes
EV density (g/ml)
1.17
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
iPSC
EV-harvesting Medium
Serum free medium
Cell count
2.00E+07
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
25
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
37432
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
20%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
11
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
150000
Duration (min)
600
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: speed (g)
150000
Pelleting: adjusted k-factor
10018
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected contaminants
GM130/ Calnexin
Flow cytometry
Type of Flow cytometry
Nano-flow Cytometry
Hardware adaptation to ~100nm EV's
By utilizing Rayleigh scattering
Calibration bead size
0.25
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Proteomics database
ProteomeXchange
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Nano-flow Cytometry
Hardware adjustment
By utilizing Rayleigh scattering
Calibration bead size
68/ 91,113,155
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.03E+08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100
EV230372 7/14 Homo sapiens H2228 (d)(U)C
DG
Schöne N 2024 100%

Study summary

Full title
All authors
Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A
Journal
J Extracell Vesicles
Abstract
Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide)
EV-METRIC
100% (99th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
large EVs
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
Density gradient
Protein markers
EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6
non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Proteomics
no
EV density (g/ml)
1.10-1.17
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H2228
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
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
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
17000
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
Heraeus 3331
Wash: speed (g)
17000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
16
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Actinin-4/ Rgap1/ Mitofilin/ Arf6/ CD81
Not detected EV-associated proteins
Syntenin
Not detected contaminants
GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Flow cytometry
Type of Flow cytometry
standard flow cytometer
Calibration bead size
0.2, 0.8
Antibody details provided?
No
Detected EV-associated proteins
EMMPRIN/ EpCAM/ EGFR
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
196.8
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 4.83E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230372 9/14 Homo sapiens H596 (d)(U)C
DG
Schöne N 2024 100%

Study summary

Full title
All authors
Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A
Journal
J Extracell Vesicles
Abstract
Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide)
EV-METRIC
100% (99th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
large EVs
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
Density gradient
Protein markers
EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6
non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Proteomics
no
EV density (g/ml)
1.10-1.17
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H596
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
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
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
17000
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
Heraeus 3331
Wash: speed (g)
17000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
16
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Actinin-4/ Rgap1/ Mitofilin/ Arf6
Not detected EV-associated proteins
Syntenin/ CD81
Not detected contaminants
GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Flow cytometry
Type of Flow cytometry
standard flow cytometer
Calibration bead size
0.2, 0.8
Antibody details provided?
No
Detected EV-associated proteins
EMMPRIN/ EpCAM/ EGFR
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
188.7
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.33E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230372 13/14 Homo sapiens Blood plasma (d)(U)C Schöne N 2024 100%

Study summary

Full title
All authors
Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A
Journal
J Extracell Vesicles
Abstract
Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide)
EV-METRIC
100% (99th 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
Blood plasma
Sample origin
NSCLC
Focus vesicles
large EVs
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: Actinin-4/ Rgap1/ EMMPRIN/ EpCAM/ EGFR
non-EV: ApoA1/ ApoB
Proteomics
no
EV density (g/ml)
1.10-1.17
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
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
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
17000
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
Heraeus 3331
Wash: speed (g)
17000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
16
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Actinin-4/ Rgap1/ EMMPRIN
Detected contaminants
ApoB
Not detected contaminants
ApoA1
Flow cytometry
Type of Flow cytometry
standard flow cytometer
Calibration bead size
0.2, 0.8
Antibody details provided?
No
Detected EV-associated proteins
EMMPRIN/ EpCAM/ EGFR
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
187
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 6.05E+08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230372 3/14 Homo sapiens HCC-78 (d)(U)C
DG
Schöne N 2024 78%

Study summary

Full title
All authors
Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A
Journal
J Extracell Vesicles
Abstract
Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide)
EV-METRIC
78% (97th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
large EVs
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
Density gradient
Protein markers
EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6
non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Proteomics
no
EV density (g/ml)
1.10-1.17
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HCC-78
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
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
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
17000
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
Heraeus 3331
Wash: speed (g)
17000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
16
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Actinin-4/ Rgap1/ Syntenin-1/ Arf6/ Mitofilin
Not detected EV-associated proteins
CD81
Not detected contaminants
GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Flow cytometry
Type of Flow cytometry
standard flow cytometer
Calibration bead size
0.2, 0.8
Antibody details provided?
No
Detected EV-associated proteins
EMMPRIN/ EpCAM/ EGFR
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
200.13
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 5.63E+09
EV230372 8/14 Homo sapiens H2228 (d)(U)C Schöne N 2024 78%

Study summary

Full title
All authors
Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A
Journal
J Extracell Vesicles
Abstract
Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide)
EV-METRIC
78% (97th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
small EVs
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: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin
non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H2228
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation 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 performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
143000
Wash: volume per pellet (ml)
1.4
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
143000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin/ Arf6/ CD81
Not detected EV-associated proteins
Actinin-4/ Rgap1/ Mitofilin
Not detected contaminants
GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
163.8
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.10E+10
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230372 10/14 Homo sapiens H596 (d)(U)C
DG
Schöne N 2024 78%

Study summary

Full title
All authors
Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A
Journal
J Extracell Vesicles
Abstract
Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide)
EV-METRIC
78% (97th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
small EVs
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
Density gradient
Protein markers
EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin
non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Proteomics
no
EV density (g/ml)
1.10-1.17
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H596
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation 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 performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
143000
Wash: volume per pellet (ml)
1.4
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
143000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
16
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin/ Actinin-4/ Arf6/ CD81
Not detected EV-associated proteins
Rgap1/ Mitofilin
Not detected contaminants
GM130/ HDAC1/ Albumin/ ApoA1/ ApoB
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
141.2
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 6.09E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220326 1/4 Homo sapiens Glioblastoma stem-like cells NCH421k (d)(U)C
DC
Lokumcu T 2024 78%

Study summary

Full title
All authors
Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V
Journal
ACS Nano
Abstract
Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide)
EV-METRIC
78% (97th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
small extracellular vesicles (sEVs)
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
Density cushion
Protein markers
EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Glioblastoma stem-like cells NCH421k
EV-harvesting Medium
Serum free medium
Cell viability (%)
91 - 93
Cell count
194200000 - 222312000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
70
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
100000
Density cushion
Density medium
Iodixanol
Sample volume
7
Cushion volume
4
Density of the cushion
20%
Centrifugation time
70
Centrifugation speed
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
Detected contaminants
60S acidic ribosomal protein P0 (RPLP0)
Not detected contaminants
GM130/ Lamin B1/ Cytochrome c
Proteomics database
ProteomeXchange
Detected contaminants
Albumin/ Calreticulin/ Calnexin
Not detected contaminants
Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
175.0
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 4,58E+12
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220326 2/4 Homo sapiens Glioblastoma stem-like cells NCH644 (d)(U)C
DC
Lokumcu T 2024 78%

Study summary

Full title
All authors
Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V
Journal
ACS Nano
Abstract
Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide)
EV-METRIC
78% (97th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
small extracellular vesicles (sEVs)
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
Density cushion
Protein markers
EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Glioblastoma stem-like cells NCH644
EV-harvesting Medium
Serum free medium
Cell viability (%)
83 - 90
Cell count
225576000 - 231070000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
70
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
100000
Density cushion
Density medium
Iodixanol
Sample volume
7
Cushion volume
4
Density of the cushion
20%
Centrifugation time
70
Centrifugation speed
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
Detected contaminants
60S acidic ribosomal protein P0
Not detected contaminants
GM130/ Lamin B1/ Cytochrome c
Proteomics database
ProteomeXchange
Detected contaminants
Albumin/ Calreticulin/ Calnexin
Not detected contaminants
Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
169.7
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1,87E+12
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220326 3/4 Homo sapiens Glioblastoma stem-like cells NCH705 (d)(U)C
DC
Lokumcu T 2024 78%

Study summary

Full title
All authors
Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V
Journal
ACS Nano
Abstract
Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide)
EV-METRIC
78% (97th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
small extracellular vesicles (sEVs)
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
Density cushion
Protein markers
EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Glioblastoma stem-like cells NCH705
EV-harvesting Medium
Serum free medium
Cell viability (%)
98
Cell count
204312000 - 225840000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
70
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
100000
Density cushion
Density medium
Iodixanol
Sample volume
7
Cushion volume
4
Density of the cushion
20%
Centrifugation time
70
Centrifugation speed
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
Detected contaminants
60S acidic ribosomal protein P0 (RPLP0)
Not detected contaminants
GM130/ Lamin B1/ Cytochrome c
Proteomics database
ProteomeXchange
Detected contaminants
Albumin/ Calreticulin/ Calnexin
Not detected contaminants
Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
179.7
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 6,05E+12
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220326 4/4 Homo sapiens Glioblastoma stem-like cells NCH711d (d)(U)C
DC
Lokumcu T 2024 78%

Study summary

Full title
All authors
Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V
Journal
ACS Nano
Abstract
Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide)
EV-METRIC
78% (97th 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
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
small extracellular vesicles (sEVs)
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
Density cushion
Protein markers
EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Glioblastoma stem-like cells NCH711d
EV-harvesting Medium
Serum free medium
Cell viability (%)
89 - 91
Cell count
102240000 - 116112000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
70
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
100000
Density cushion
Density medium
Iodixanol
Sample volume
7
Cushion volume
4
Density of the cushion
20%
Centrifugation time
70
Centrifugation speed
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1
Detected contaminants
60S acidic ribosomal protein P0 (RPLP0)
Not detected contaminants
GM130/ Lamin B1/ Cytochrome c
Proteomics database
ProteomeXchange
Detected contaminants
Albumin/ Calreticulin/ Calnexin
Not detected contaminants
Argonaute 2/ GM130/ PMP70/ Tamm-Horsfall protein/ Cytochrome c1
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
203.5
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2,87E+12
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230995 1/6 Homo sapiens MML-1 (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% (94th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Flotillin-1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MML-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
94
Cell count
50000
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
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ Flotillin-1
Detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 761800000
EM
EM-type
Transmission-EM
Image type
Close-up
EV230995 2/6 Homo sapiens UM22Bap1+/+ (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% (94th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Flotillin-1
non-EV: Calnexin/ bovine HBA1c/ bovine CPN1
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
UM22Bap1+/+
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
94
Cell count
50000
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
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ Flotillin-1
Not detected contaminants
Calnexin
ELISA
Antibody details provided?
No
Detected contaminants
bovine HBA1c/ bovine CPN1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 936666666.7
EM
EM-type
Transmission-EM
Image type
Close-up
EV230995 3/6 Homo sapiens UM22Bap1-/- (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% (94th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Flotillin-1/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
UM22Bap1-/-
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
93
Cell count
50000
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
Antibody details provided?
No
Detected EV-associated proteins
CD81/ Flotillin-1
Not detected EV-associated proteins
CD63
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 36277777.78
EM
EM-type
Transmission-EM
Image type
Close-up
EV230995 4/6 Homo sapiens MML-1 (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% (94th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Flotillin-1
non-EV: Calnexin/ bovine CPN1
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MML-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
94
Cell count
50000
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
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ Flotillin-1
Not detected contaminants
Calnexin
ELISA
Antibody details provided?
No
Detected contaminants
bovine CPN1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 139727777.8
EM
EM-type
Transmission-EM
Image type
Close-up
EV230995 5/6 Homo sapiens UM22Bap1+/+ (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% (94th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Flotillin-1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
UM22Bap1+/+
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
94
Cell count
50000
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
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ Flotillin-1
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 82011111.11
EM
EM-type
Transmission-EM
Image type
Close-up
EV230995 6/6 Homo sapiens UM22Bap1-/- (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% (94th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Flotillin-1
non-EV: Calnexin/ bovine CPN1
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
UM22Bap1-/-
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
93
Cell count
50000
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
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ Flotillin-1
Not detected contaminants
Calnexin
ELISA
Antibody details provided?
No
Detected contaminants
bovine CPN1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 342605555.6
EM
EM-type
Transmission-EM
Image type
Close-up
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
Antibody details provided?
No
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
Antibody details provided?
No
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
Antibody details provided?
No
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
EV230572 3/6 Homo sapiens HEK293-GFP (d)(U)C Djeungoue-Petga, Marie-Ange 2024 67%

Study summary

Full title
All authors
Marie Ange Djeungoue Petgaa, Catherine Taylora, Alexander Macpherson, Surendar Reddy Dhadi, Thomas Rollin, Jeremy W. Roya, Anirban Ghosh, Stephen M. Lewis, Rodney J. Ouellette
Journal
Abstract
Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular (show more...)Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular delivery of therapeutic cargo. Precipitation-based methods for the isolation of EVs remain popular due to ease of use and lack of requirements for specialized equipment. We describe here a novel charge-based EV isolation method that is simple, scalable, and uses inexpensive polyethylenimine (PEI) polymers. GFP-expressing EVs were isolated from the conditioned cell culture (CCM) media of HEK293-GFP cells using either branched 10 kDa PEI (B-PEI) or linear 25 kDa PEI (L-PEI). Isolated EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy (TEM), and flow cytometry. Western blotting for common EV markers, including CD63, CD9, flotillin-1, and heat shock protein 70 were positive, while GRP94, a marker for cellular contamination, was negative. Isolated EVs had a mean diameter of 146 nm for B-PEI and 175 nm for L- PEI, while TEM revealed a spherical cup-shaped appearance typical of EVs. In addition, we determined that PEI-based EV isolation methods were scalable up to volumes of at least 50 mL. EVs isolated from CCM collected from SUM159 cells that express CD63 fused to a dual EGFP-Renilla-split tag were tested for their ability to reconstitute functional luciferase by delivering the CD63-EGFP-Renilla-split tag to SUM159 recipient cells loaded with a cytopermeable Renilla luciferase substrate. Although EVs isolated using L-PEI behaved similarly to EVs isolated using ultracentrifugation, we observed that EVs isolated using B-PEI produced a more rapid uptake and delivery of active luciferase. In this study we demonstrate that both branched and linear PEI polymers can precipitate EVs from CCM. Furthermore, once eluted from the polymers, the isolated EVs were able to deliver functional protein cargo to recipient cells. Overall, our data support PEI-based isolation of EVs as a simple, rapid method for the recovery of functional EVs. (hide)
EV-METRIC
67% (94th 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
Cell culture supernatant
Sample origin
GFP overexpression
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: CD9/ CD63/ Flotillin-1/ HSP70/ GFP
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Mechanism of uptake/transfer/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HEK293-GFP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Flotillin-1/ HSP70/ GFP
Not detected contaminants
GRP94
Flow cytometry
Type of Flow cytometry
Beckman Coulter Cytoflex
Hardware adaptation to ~100nm EV's
The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells)
Calibration bead size
0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9
Antibody details provided?
No
Detected EV-associated proteins
GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
~172
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 6.00E+09
Particle analysis: flow cytometry
Flow cytometer type
Nanoscale
Hardware adjustment
The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells)
Calibration bead size
0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 9.00E+06
EM
EM-type
Transmission-EM
Image type
Close-up
EV240036 5/6 Homo sapiens Serum Exoquick
IAF
Shinde U 2024 63%

Study summary

Full title
All authors
Shinde U, Rao A, Bansal V, Das DK, Balasinor NH, Madan T
Journal
Reproduction
Abstract
Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic (show more...)Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. (hide)
EV-METRIC
63% (94th 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
Pregnant
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
Commercial method
Immunoaffinity capture (non-commercial)
Protein markers
EV: CD9/ CD63/ PLAP/ Cullin 7
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
Commercial kit
Exoquick
Immunoaffinity capture
Selected surface protein(s)
PLAP
EV-subtype
Distinction between multiple subtypes
affinity capture
Used subtypes
PLAP+
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ PLAP/ Cullin 7
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
RT(q)PCR
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNAse
RNAse concentration
20mg/mL
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
30-150 nm
NTA
Report type
Modus
Reported size (nm)
30-150 nm
EV concentration
Yes
Particle yield
number of particles per million cells: 1E10 particles/ml
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
30-150 nm
EV230572 4/6 Homo sapiens HEK293-GFP PEI precipitation Djeungoue-Petga, Marie-Ange 2024 63%

Study summary

Full title
All authors
Marie Ange Djeungoue Petgaa, Catherine Taylora, Alexander Macpherson, Surendar Reddy Dhadi, Thomas Rollin, Jeremy W. Roya, Anirban Ghosh, Stephen M. Lewis, Rodney J. Ouellette
Journal
Abstract
Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular (show more...)Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular delivery of therapeutic cargo. Precipitation-based methods for the isolation of EVs remain popular due to ease of use and lack of requirements for specialized equipment. We describe here a novel charge-based EV isolation method that is simple, scalable, and uses inexpensive polyethylenimine (PEI) polymers. GFP-expressing EVs were isolated from the conditioned cell culture (CCM) media of HEK293-GFP cells using either branched 10 kDa PEI (B-PEI) or linear 25 kDa PEI (L-PEI). Isolated EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy (TEM), and flow cytometry. Western blotting for common EV markers, including CD63, CD9, flotillin-1, and heat shock protein 70 were positive, while GRP94, a marker for cellular contamination, was negative. Isolated EVs had a mean diameter of 146 nm for B-PEI and 175 nm for L- PEI, while TEM revealed a spherical cup-shaped appearance typical of EVs. In addition, we determined that PEI-based EV isolation methods were scalable up to volumes of at least 50 mL. EVs isolated from CCM collected from SUM159 cells that express CD63 fused to a dual EGFP-Renilla-split tag were tested for their ability to reconstitute functional luciferase by delivering the CD63-EGFP-Renilla-split tag to SUM159 recipient cells loaded with a cytopermeable Renilla luciferase substrate. Although EVs isolated using L-PEI behaved similarly to EVs isolated using ultracentrifugation, we observed that EVs isolated using B-PEI produced a more rapid uptake and delivery of active luciferase. In this study we demonstrate that both branched and linear PEI polymers can precipitate EVs from CCM. Furthermore, once eluted from the polymers, the isolated EVs were able to deliver functional protein cargo to recipient cells. Overall, our data support PEI-based isolation of EVs as a simple, rapid method for the recovery of functional EVs. (hide)
EV-METRIC
63% (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
Cell culture supernatant
Sample origin
GFP overexpresion
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
PEI precipitation
Protein markers
EV: CD9/ CD63/ Flotillin-1/ HSP70/ GFP
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Mechanism of uptake/transfer/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HEK293-GFP
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Separation Method
Other
Name other separation method
PEI precipitation
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Flotillin-1/ HSP70/ GFP
Not detected contaminants
GRP94
Flow cytometry
Type of Flow cytometry
Beckman Coulter Cytoflex
Hardware adaptation to ~100nm EV's
The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells)
Calibration bead size
0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9
Antibody details provided?
No
Detected EV-associated proteins
GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
146-200
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1-6E10
Particle analysis: flow cytometry
Flow cytometer type
Nanoscale
Hardware adjustment
The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells)
Calibration bead size
0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9
Particle yield
particles per milliliter of starting sample: 1-6E10
EM
EM-type
Transmission-EM
Image type
Close-up
EV230012 1/4 Mus musculus cardiac mesenchymal stromal cells Filtration
UF
SEC (non-commercial)
Caller T 2024 63%

Study summary

Full title
All authors
Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J
Journal
Circulation
Abstract
Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide)
EV-METRIC
63% (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
Cell culture supernatant
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
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
cardiac mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
90
Cell count
3000000
Separation Method
Filtration steps
0.8
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Cellulose acetate
Size-exclusion chromatography
Total column volume (mL)
70
Sample volume/column (mL)
10
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ TSG101
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
>250
EV concentration
Yes
Particle yield
number of particles per million cells: 90000000
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV230012 2/4 Mus musculus cardiac mesenchymal stromal cells Filtration
UF
SEC (non-commercial)
Caller T 2024 63%

Study summary

Full title
All authors
Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J
Journal
Circulation
Abstract
Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide)
EV-METRIC
63% (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
Cell culture supernatant
Sample origin
Heart failure
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
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
cardiac mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
90
Cell count
3000000
Separation Method
Filtration steps
0.8
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Cellulose acetate
Size-exclusion chromatography
Total column volume (mL)
70
Sample volume/column (mL)
10
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ TSG101
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
<250
EV concentration
Yes
Particle yield
number of particles per million cells: 40000000
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV230012 3/4 Mus musculus Cardiac tissue Filtration
UF
SEC (non-commercial)
Caller T 2024 63%

Study summary

Full title
All authors
Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J
Journal
Circulation
Abstract
Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide)
EV-METRIC
63% (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
Cardiac tissue
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
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cardiac tissue
Separation Method
Filtration steps
0.8
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Cellulose acetate
Size-exclusion chromatography
Total column volume (mL)
70
Sample volume/column (mL)
10
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ TSG101
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
<250
EV concentration
Yes
Particle yield
number of particles per 100mg tissue: 90000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230012 4/4 Mus musculus Cardiac tissue Filtration
UF
SEC (non-commercial)
Caller T 2024 63%

Study summary

Full title
All authors
Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J
Journal
Circulation
Abstract
Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide)
EV-METRIC
63% (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
Cardiac tissue
Sample origin
Heart failure
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
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cardiac tissue
Separation Method
Filtration steps
0.8
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Cellulose acetate
Size-exclusion chromatography
Total column volume (mL)
70
Sample volume/column (mL)
10
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ TSG101
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
<250
EV concentration
Yes
Particle yield
number of particles per 100 mg tissue: 190000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210336 1/2 Homo sapiens THP1 (d)(U)C
DG
Driedonks, Tom 2024 63%

Study summary

Full title
All authors
Tom A. P. Driedonks, Sarah Ressel, Thi Tran Ngoc Minh, Amy H. Buck, Esther N. M. Nolte-‘t Hoen
Journal
J Extracell Biol
Abstract
Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-siz (show more...)Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners. (hide)
EV-METRIC
63% (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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: CD9/ CD63/ CD81
non-EV: None
Proteomics
no
EV density (g/ml)
1.11 -1.18
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
THP1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.0 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.2
Orientation
Bottom-up
Speed (g)
192000
Duration (min)
900-1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: speed (g)
192000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
according to van der Vlist et al, Nature Protocols 2012
Calibration bead size
0.1/ 0.2
EV concentration
Yes
EV210336 2/2 Homo sapiens THP1 (d)(U)C
DG
Driedonks, Tom 2024 63%

Study summary

Full title
All authors
Tom A. P. Driedonks, Sarah Ressel, Thi Tran Ngoc Minh, Amy H. Buck, Esther N. M. Nolte-‘t Hoen
Journal
J Extracell Biol
Abstract
Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-siz (show more...)Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners. (hide)
EV-METRIC
63% (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
Cell culture supernatant
Sample origin
Pam3CSK4 treated
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
Density gradient
Protein markers
EV: CD9/ CD63/ CD81
non-EV: None
Proteomics
no
EV density (g/ml)
1.11 -1.18
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
THP1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.0 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.2
Orientation
Bottom-up
Speed (g)
192000
Duration (min)
900-1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: speed (g)
192000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
according to van der Vlist et al Nature Protocols 2012
Calibration bead size
0.1/ 0.2
EV concentration
Yes
EV230597 3/9 Homo sapiens 293T (d)(U)C Chen Z 2024 56%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
56% (90th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ CD63/ TSG101/ CD81
non-EV: GM130/ Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
293T
EV-harvesting Medium
Serum free medium
Cell count
1.00E+07
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
25
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected contaminants
GM130/ Calnexin
Flow cytometry
Type of Flow cytometry
Nano-flow Cytometry
Hardware adaptation to ~100nm EV's
By utilizing Rayleigh scattering
Calibration bead size
0.25
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Nano-flow Cytometry
Hardware adjustment
By utilizing Rayleigh scattering
Calibration bead size
68/ 91,113,155
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.20E+08
EV230597 4/9 Homo sapiens huMSC (d)(U)C Chen Z 2024 56%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
56% (90th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD63/ TSG101/ CD81
non-EV: GM130/ Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
huMSC
EV-harvesting Medium
Serum free medium
Cell count
5.00E+06
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
25
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected contaminants
GM130/ Calnexin
Flow cytometry
Type of Flow cytometry
Nano-flow Cytometry
Hardware adaptation to ~100nm EV's
By utilizing Rayleigh scattering
Calibration bead size
0.25
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Nano-flow Cytometry
Hardware adjustment
By utilizing Rayleigh scattering
Calibration bead size
68/ 91/ 113/ 155
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 7.71E+07
EV230597 5/9 Homo sapiens HFF-1 (d)(U)C Chen Z 2024 56%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
56% (90th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ CD63/ TSG101/ CD81
non-EV: GM130/ Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HFF-1
EV-harvesting Medium
Serum free medium
Cell count
1.00E+07
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
25
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected contaminants
GM130/ Calnexin
Flow cytometry
Type of Flow cytometry
Nano-flow Cytometry
Hardware adaptation to ~100nm EV's
By utilizing Rayleigh scattering
Calibration bead size
0.25
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Nano-flow Cytometry
Hardware adjustment
By utilizing Rayleigh scattering
Calibration bead size
68/ 91/ 113/ 155
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 8.57E+07
EV230597 6/9 Homo sapiens A549 (d)(U)C Chen Z 2024 56%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
56% (90th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ CD63/ TSG101/ CD81
non-EV: GM130/ Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
A549
EV-harvesting Medium
Serum free medium
Cell count
1.00E+07
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
25
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected contaminants
GM130/ Calnexin
Flow cytometry
Type of Flow cytometry
Nano-flow Cytometry
Hardware adaptation to ~100nm EV's
By utilizing Rayleigh scattering
Calibration bead size
0.25
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Nano-flow Cytometry
Hardware adjustment
By utilizing Rayleigh scattering
Calibration bead size
68/ 91/ 113/ 155
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 9.78E+07
EV230597 7/9 Homo sapiens H460 (d)(U)C Chen Z 2024 56%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
56% (90th 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
Cell culture supernatant
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
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ CD63/ TSG101/ CD81
non-EV: GM130/ Calnexin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H460
EV-harvesting Medium
Serum free medium
Cell count
1.00E+07
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
25
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected contaminants
GM130/ Calnexin
Flow cytometry
Type of Flow cytometry
Nano-flow Cytometry
Hardware adaptation to ~100nm EV's
By utilizing Rayleigh scattering
Calibration bead size
0.25
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Nano-flow Cytometry
Hardware adjustment
By utilizing Rayleigh scattering
Calibration bead size
68/ 91/ 113/ 155
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.44E+08
EV230597 8/9 Homo sapiens MCF7 (d)(U)C Chen Z 2024 56%

Study summary

Full title
All authors
Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y
Journal
J Extracell Vesicles
Abstract
Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide)
EV-METRIC
56% (90th 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