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Exosomes and EVs characterization

Size, concentration, count, phenotyping, cargo, etc.

Alfatestlab is equipped with the latest technology platform to provide rapid, reliable and accurate characterization of EVs and exosomes following MISEV 2018 guidelines1. Based on NTA and Exoview analysis, our services for EVs and exosomes offers multidimensional data sets enabling an advanced understanding of complex EV populations. Services include: Sizing, Concentration per size range, Membrane labelling, Count, Phenotyping, Cargo, Integrity, Purity.

1 Thery, C., et al. Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 7:1535750 (2018)

Instruments ExoView® (NanoView Biosciences), NanoSight NS300 (Malvern Panalytical)
Samples Particles, Biomolecules
Industry Biotech

Alfatestlab offers Basic analysis package as well as Advanced analysis package, in any case we provide

Guaranted analysis upon delivery2

Upon agreement on sample shipment date

  •  Basic analysis package includes:

EVs and exosome concentration3

Alfatestlab will provide the concentration of your EVs sample expressed in number of particles per mL, as a function of the hydrodynamic diameter i.e. the number of particles per volume for any size range.
Each analysis is performed in 5 replicates, and hydrodynamic diameter parameters provided are: mode, mean, d10, d50, d90, standard deviation. This analysis is extremely useful to compare exosomes samples and evaluate the relative number of particles in a specific size range (30-130nm / 130-200nm, etc.) Moreover, these concentration data expressed in number of particles/mL can be compared with ELISA data expressed in protein/mL
3 This analysis requires purified samples

Membrane staining

EVs and exosome concentration data can be implemented with DIO, DII or Cell Mask Orange fluorescence labellling in order to segregate data from lipidic species respect to the rest of the mixture components.
  

  

  

  • Advanced analysis package provides multiparametric characterization of single EVs

Count, size and phenotyping

This analysis requires NO PURIFICATION, cell culture supernatant, plasma, serum, urine, and other matrices can be measured directly. Alfatestlab will provide size (<50nm) and count data of your entire sample expressed in number of particles per size range. Alfatestlab provides protein quantification and protein colocalization services thanks to antigen-specific capture.
 

Standard membrane protein profile of individual EVs is provided based on ubiquitous Tetraspanin markers CD9, CD63, and CD81 (CD41a option). Custom staining for analysis of other proteins of interest is available upon request.

  

Purity: validation of your exosome isolation method

By comparing the quantity of EVs in crude biological fluid vs. isolated/purified sample Alfatestlab provides the perfect tool to validate your purification methods from cell culture with or without bovine EVs, blood plasma, blood serum, cerebrospinal fluid (CSF), saliva, urine, follicular fluid, synovial fluid, etc.

  

Cargo and integrity of EVs

At Alfatestlab we know that understanding how efficiently your therapeutics are loaded into EVs and how efficiently they are purified is a crucial information.  Alfatestlab can provide measurement of both external and internal proteins at the single-vesicle level. 2018 MISEV guidelines recommend intracellular cytosolic protein detection as part of standard protocols for EV characterization. For this purpose, we provide measurement of intracellular cytosolic proteins, including ALIX and Syntenin. EVs are captured, incubated with up to three fluorescent antibodies or dyes, which can be targeted against surface, and then permeabilized to enable detection of luminal proteins or target specific antibodies to EV cargo. EV subpopulation fractions can be then quantified in terms of the number of vesicles containing specific cargo proteins.

 

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