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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 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
EV190090 1/1 Homo sapiens Cell culture supernatant (d)(U)C
Torres Crigna A 2019 67%

Study summary

Full title
All authors
Torres Crigna A., Fricke F., Nitschke K., Worst T., Erb U., Karremann M., Buschmann D., Elvers-Hornung S., Tucher C., Schiller M., Hausser I., Gebert J., Bieback K.
Transfus Med Hemother
Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioacti (show more...)Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation. Methods: Conditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Results: EVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield. Conclusions: Our findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies. (hide)
67% (92nd percentile of all experiments on the same sample type)
 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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Protein markers
EV: TSG101/ CD81/ CD63/ CD9/ Alix
non-EV: calnexin
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Cell viability (%)
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 :
Pelleting: time(min)
Pelleting: rotor type
Surespin 630 (17 ml)
Pelleting: speed (g)
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix/ CD81
Not detected contaminants
Flow cytometry specific beads
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected EV-associated proteins
Not detected contaminants
Image type
Report size (nm)
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  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
Homo sapiens
sample type
Cell culture
cell type
Control condition
separation protocol
Exp. nr.