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You searched for: EV190105 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190105 1/2 Homo sapiens Cell culture supernatant DG
(d)(U)C
Emanuela Mensà 2020 100%

Study summary

Full title
All authors
Emanuela Mensà, Michele Guescini, Angelica Giuliani, Maria Giulia Bacalini, Deborah Ramini, Giacomo Corleone, Manuela Ferracin, Gianluca Fulgenzi, Laura Graciotti, Francesco Prattichizzo, Leonardo Sorci, Michela Battistelli, Vladia Monsurrò, Anna Rita Bonfigli, Maurizio Cardelli, Rina Recchioni, Fiorella Marcheselli, Silvia Latini, Serena Maggio, Mirco Fanelli, Stefano Amatori, Gianluca Storci, Antonio Ceriello, Vilberto Stocchi, Maria De Luca, Luca Magnani, Maria Rita Rippo, Antonio Domenico Procopio, Claudia Sala, Iva Budimir, Cristian Bassi, Massimo Negrini, Paolo Garagnani, Claudio Franceschi, Jacopo Sabbatinelli, Massimiliano Bonafè, Fabiola Olivieri
Journal
J Extracell Vesicles
Abstract
The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. H (show more...)The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication. (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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C
Protein markers
EV: TSG101/ Calnexin/ CD63
non-EV: Albumin
Proteomics
no
EV density (g/ml)
1.08-1.10
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Human umbilical vein endothelial cells (HUVECs)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
103745
Wash: volume per pellet (ml)
3
Wash: time (min)
70
Wash: Rotor Type
Type 90 Ti
Wash: speed (g)
109354
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.5
Orientation
Bottom-up
Rotor type
SW 28
Speed (g)
103745
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
3.5
Pelleting: duration (min)
70
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
109354
Pelleting-wash: volume per pellet (mL)
3
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
Type 90 Ti
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ Calnexin
Not detected contaminants
Albumin
Characterization: RNA analysis
Proteinase treatment
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
20
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
80 (sEVs) / 120 (lEVs)
EV concentration
Yes
Particle yield
Number of particles per cell: 3E4 (sEVs)/1.5E4 (lEVs)
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190105 2/2 Homo sapiens Cell culture supernatant (d)(U)C Emanuela Mensà 2020 43%

Study summary

Full title
All authors
Emanuela Mensà, Michele Guescini, Angelica Giuliani, Maria Giulia Bacalini, Deborah Ramini, Giacomo Corleone, Manuela Ferracin, Gianluca Fulgenzi, Laura Graciotti, Francesco Prattichizzo, Leonardo Sorci, Michela Battistelli, Vladia Monsurrò, Anna Rita Bonfigli, Maurizio Cardelli, Rina Recchioni, Fiorella Marcheselli, Silvia Latini, Serena Maggio, Mirco Fanelli, Stefano Amatori, Gianluca Storci, Antonio Ceriello, Vilberto Stocchi, Maria De Luca, Luca Magnani, Maria Rita Rippo, Antonio Domenico Procopio, Claudia Sala, Iva Budimir, Cristian Bassi, Massimo Negrini, Paolo Garagnani, Claudio Franceschi, Jacopo Sabbatinelli, Massimiliano Bonafè, Fabiola Olivieri
Journal
J Extracell Vesicles
Abstract
The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. H (show more...)The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication. (hide)
EV-METRIC
43% (71st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Replicative senescence
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Replicative senescence
EV-producing cells
Human umbilical vein endothelial cells (HUVECs)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
103745
Wash: volume per pellet (ml)
3
Wash: time (min)
70
Wash: Rotor Type
Type 90 Ti
Wash: speed (g)
109354
EV-subtype
Distinction between multiple subtypes
Size
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Not detected contaminants
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
80 (sEVs) / 130 (lEVs)
EV concentration
Yes
Particle yield
Number of particles per cell: 1.3E5 (sEVs)/1.5E4 (lEVs)
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
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