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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
EV200058 1/1 Homo sapiens Cell culture supernatant (d)(U)C
DG
Luis A Arteaga-Blanco 2020 100%

Study summary

Full title
All authors
Luis A Arteaga-Blanco, Andrés Mojoli, Robson Q Monteiro, Vanessa Sandim, Rubem F S Menna-Barreto, Filipe Santos Pereira-Dutra, Patrícia T Bozza, Rafael de Oliveira Resende, Dumith Chequer Bou-Habib
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of t (show more...)Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes. (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
Other / Small EVs
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 + DG
Protein markers
EV: Alix/ CD81/ CD63/ beta actin
non-EV: Calnexin/ Cytochrome c
Proteomics
no
EV density (g/ml)
1.117- 1.181
Show all info
Study aim
Biomarker/protocol adaptation for the isolation and characterization of MDM-derived small EVs
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary human macrophages derived from circulating monocytes
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
130
Wash: volume per pellet (ml)
10
Wash: time (min)
70
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
130 000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Continuous
Lowest density fraction
10%
Highest density fraction
90%
Total gradient volume, incl. sample (mL)
11
Sample volume (mL)
0,2
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: duration (min)
70
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
130
Pelleting-wash: volume per pellet (mL)
10
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
SW 41 Ti
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD63/ beta actin/ Alix/ CD81
Not detected contaminants
Calnexin/ Cytochrome c
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
Report size (nm)
80
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