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You searched for: EV210165 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210165 1/2 Homo sapiens THP-1 DG
DC
Phu TA 2022 75%

Study summary

Full title
All authors
Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL
Journal
Mol Ther
Abstract
Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARĪ³)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide)
EV-METRIC
75% (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
exosome
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
Density gradient
Density cushion
Protein markers
EV: CD81/ CD63/ CD9
non-EV: Calnexin/ GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
1,00E+06
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Density cushion
Density medium
Iodixanol
Sample volume
98
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected contaminants
Calnexin/ GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0,4
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
as number of particles per million cells: 6.00e+0
EV210165 2/2 Homo sapiens THP-1 DG
DC
Phu TA 2022 75%

Study summary

Full title
All authors
Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL
Journal
Mol Ther
Abstract
Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARĪ³)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide)
EV-METRIC
75% (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
IL-4 cytokine treated
Focus vesicles
exosome
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
Density gradient
Density cushion
Protein markers
EV: CD81/ CD63/ CD9
non-EV: Calnexin/ GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
1,00E+06
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Density cushion
Density medium
Iodixanol
Sample volume
98
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected contaminants
Calnexin/ GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0,4
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
90,5
EV concentration
Yes
Particle yield
as number of particles per million cells: 5.00e+0
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210165
species
Homo sapiens
sample type
Cell culture
cell type
THP-1
condition
Control condition
IL-4
cytokine treated
separation protocol
Density gradient/ DC
Density gradient/ DC
Exp. nr.
1
2
EV-METRIC %
75
75