The quantification and characterization of EVs are valuable not only for elucidating the biogenesis and cargo sorting of EVs, but also for identifying physiological and pathological markers. Creative Biolabs offers a comprehensive EV characterization and quantification service to support the understanding of the properties and downstream applications of EVs.

Since no single method can accurately phenotype, size and enumerate all EVs, thus providing all the necessary information to truly understand the biology of EVs, Creative Biolabs offers a combination of optional methods for the characterization and quantification of EVs, including but not limited to.

• Nanoparticle Tracking Analysis (NTA): The technique has been recognized by the EVs research field as one of the characterization tools. The Brownian motion of each particle is tracked and analyzed, and the hydrodynamic diameter and concentration of EVs are calculated by combining the Stockes-Einstein equation. Compared to other characterization methods, NTA offers simpler sample handling, better assurance of the original state of EVs, and faster detection. It is also suitable for the characterization of samples such as liposomes, proteins, and biological nanomaterials.
• Transmission electron microscopy (TEM): After negative staining of EVs with uranyl acetate and cellulose, the surface features of EVs can be observed by TEM. In the images, these EVs can be clearly distinguished into different sizes.
• Fluorescence activated Cell Sorting (FACS): Micron-sized latex beads are used to bind EVs. The bound EVs are then stained with fluorescent antibodies and their protein markers are identified. The EVs are ejected in a single row by the nozzle of the flow chamber under the constraint of the sheath fluid, forming a column of EVs, the latter intersecting perpendicularly with the incident laser beam, and the EVs in the column are excited by the laser to produce fluorescence. The optical system is collecting signals such as fluorescence, light scattering, light absorption or electrical impedance and feeding back to the computer system for statistical analysis of EVs characterization.
• Western Blotting: The most common technique used in EV protein assessment to suggest the presence of target proteins associated with EVs. During the procedure, purified EVs are treated with a buffered lysate containing denaturant and protease inhibitors. The protein lysates are then separated by electrophoresis and transferred to membranes for immunoblotting of specific protein targets. The approach can provide useful information about the size of the protein molecule.
• Enzyme-linked immunosorbent assay (ELISA): Another established technique for protein quantification that can be used in a number of different assay formats. Purified EVs or EVs lysates can be applied directly to an adsorbent column pretreated with immobilized capture antibodies. The captured EVs are used for another detection antibody. This improves the specificity of the assay for non-interacting antibodies. The ELISA is more rapid and can be used for high-throughput measurements.
• Tunable Resistive Pulse Sensing (TRPS): TRPS is available as an alternative to NTA for measuring EV concentration and size distribution. The technique is based on the nanoscale Coulter principle. It detects transient changes in ion flow that are generated by the transport of vesicles through size-tunable nanopores in a polyurethane membrane.

Fig.1 Overview of EV characterization techniques and their capabilities. (Buschmann, 2021)

For most available technologies, accurate measurement of EVs is challenged by size heterogeneity, low refractive index and lack of dynamic measurement range. With our innovative EVs platform, Creative Biolabs provides not only characterization and quantification of EVs, but also in-depth technical support from project planning to data interpretation. Please feel free to contact us to create a custom quote.

Reference

1. Buschmann, D.; et al. Separation, characterization, and standardization of extracellular vesicles for drug delivery applications. Adv Drug Deliv Rev. 2021, 174: 348-368.

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