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You searched for: EV220127 (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
EV220127 1/2 Homo sapiens lung tissue (d)(U)C
DG
Filtration
Liu, Bowen/ Jin, Yuan 2022 100%

Study summary

Full title
All authors
Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide)
EV-METRIC
100% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
lung 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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
20553 (pelleting) / 17842 (washing)
Protein markers
EV: Alix/ CD9/ CD81
non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.1
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
lung tissue
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: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
20553
Wash: volume per pellet (ml)
1.5
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
110,000
Wash: adjusted k-factor
17842
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
0.25 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.45
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
180,000
Duration (min)
780
Fraction volume (mL)
0.45
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
17842
Pelleting-wash: volume per pellet (mL)
1.5
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
TLA-55
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
0.8
Western Blot
Detected EV-associated proteins
Alix/ CD9/ CD81
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
131.7
Particle analysis: flow cytometry
Flow cytometer type
BD LSRFortessa
Hardware adjustment
use calibration beads
Calibration bead size
0.05/ 0.1/ 0.2/ 0.3/ 0.5
Report type
Size range/distribution
Reported size (nm)
100 - 200
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV220127 2/2 Mus musculus lung tissue (d)(U)C
DG
Filtration
Liu, Bowen/ Jin, Yuan 2022 100%

Study summary

Full title
All authors
Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide)
EV-METRIC
100% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
lung 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
(Differential) (ultra)centrifugation
Density gradient
Filtration
Adj. k-factor
20553 (pelleting) / 17842 (washing)
Protein markers
EV: Alix/ CD9/ Flotillin-1/ TSG101
non-EV: GM130/ Calnexin/ Albumin/ Argonaute-2/ Calreticulin/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.1
Show all info
Study aim
Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
lung tissue
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: time(min)
70
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
20553
Wash: volume per pellet (ml)
1.5
Wash: time (min)
70
Wash: Rotor Type
TLA-55
Wash: speed (g)
110,000
Wash: adjusted k-factor
17842
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
0.25 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.45
Orientation
Bottom-up
Rotor type
MLS-50
Speed (g)
180,000
Duration (min)
780
Fraction volume (mL)
0.45
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: speed (g)
110,000
Pelleting: adjusted k-factor
17842
Pelleting-wash: volume per pellet (mL)
1.5
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
TLA-55
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
0.7
Western Blot
Detected EV-associated proteins
Alix/ CD9/ Flotillin-1/ TSG101
Not detected contaminants
GM130/ Calnexin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
124.9
Particle analysis: flow cytometry
Flow cytometer type
BD LSRFortessa
Hardware adjustment
use calibration beads
Calibration bead size
0.05/ 0.1/ 0.2/ 0.3/ 0.5
Report type
Size range/distribution
Reported size (nm)
100 - 200
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV220127
species
Homo sapiens
Mus musculus
sample type
lung tissue
lung tissue
condition
Control condition
Control condition
separation protocol
dUC/
Density gradient/ Filtration
dUC/
Density gradient/ Filtration
Exp. nr.
1
2
EV-METRIC %
100
100