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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.
    • dUC = (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
EV100040 1/1 Mus musculus Cell culture supernatant DG
dUC
Filtration
Giri PK 2010 22%

Study summary

Full title
All authors
Giri PK, Kruh NA, Dobos KM, Schorey JS
Journal
Proteomics
Abstract
Exosomes are small 30-100 nm membrane vesicles released from hematopoietic and nonhematopoietic cell (show more...)Exosomes are small 30-100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC-MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis-infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP-treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis-infected cells. The exosomes from CFP-treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine. (hide)
EV-METRIC
22% (48th 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
DNF
Focus vesicles
exosomes
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
DG + dUC + Filtration
Protein markers
EV: LAMP1
non-EV:
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
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: time(min)
60
Density gradient
Density medium
Sucrose
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
LAMP1
ELISA
Detected EV-associated proteins
LAMP1
NA
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