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You searched for: EV200185 (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
EV200185 1/3 Staphylococcus aureus Cell culture supernatant DG
(d)(U)C
Filtration
Bitto, Natalie J. 2021 78%

Study summary

Full title
All authors
Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos
Journal
J Extracell Vesicles
Abstract
Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide)
EV-METRIC
78% (96th 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
Cell Name
S. aureus NCTC6571
Sample origin
Control condition
Focus vesicles
Other / Membrane vesicles (MVs)
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
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: S. aureus proteins
non-EV: None
Proteomics
no
EV density (g/ml)
1.069-1.119
Show all info
Study aim
Function
Sample
Species
Staphylococcus aureus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
S. aureus NCTC6571
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
P28S
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12ml
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
120
Pelleting-wash: speed (g)
SW 40 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
S. aureus proteins
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle yield
particles per colony forming units (CFU) of bacteria;Yes, other: 1010000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200185 2/3 Staphylococcus aureus Cell culture supernatant DG
(d)(U)C
Filtration
Bitto, Natalie J. 2021 78%

Study summary

Full title
All authors
Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos
Journal
J Extracell Vesicles
Abstract
Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide)
EV-METRIC
78% (96th 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
Cell Name
S. aureus BPH2760
Sample origin
Control condition
Focus vesicles
Other / Membrane vesicles (MVs)
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
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: S. aureus proteins
non-EV: None
Proteomics
no
EV density (g/ml)
1.069-1.119
Show all info
Study aim
Function
Sample
Species
Staphylococcus aureus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
S. aureus BPH2760
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
P28S
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12ml
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
120
Pelleting-wash: speed (g)
SW 40 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
S. aureus proteins
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle yield
particles per colony forming units (CFU) of bacteria;Yes, other: 28000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200185 3/3 Staphylococcus aureus Cell culture supernatant DG
(d)(U)C
Filtration
Bitto, Natalie J. 2021 78%

Study summary

Full title
All authors
Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos
Journal
J Extracell Vesicles
Abstract
Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide)
EV-METRIC
78% (96th 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
Cell Name
S. aureus BPH2900
Sample origin
Control condition
Focus vesicles
Other / Membrane vesicles (MVs)
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
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: S. aureus proteins
non-EV: None
Proteomics
no
EV density (g/ml)
1.069-1.119
Show all info
Study aim
Function
Sample
Species
Staphylococcus aureus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
S. aureus BPH2900
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
P28S
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12ml
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
120
Pelleting-wash: speed (g)
SW 40 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
S. aureus proteins
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle yield
particles per colony forming units (CFU) of bacteria;Yes, other: 53000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
1 - 3 of 3
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200185
species
Staphylococcus
aureus
sample type
Cell culture
cell type
S. aureus NCTC6571
S. aureus BPH2760
S. aureus BPH2900
condition
Control condition
Control condition
Control condition
separation protocol
Density gradient
dUC
Filtration
Density gradient
dUC
Filtration
Density gradient
dUC
Filtration
Exp. nr.
1
2
3
EV-METRIC %
78
78
78