Extracellular vehicles (EVs) are lipid bound vesicles secreted by cells into the extracellular space. The three main subtypes of EVs are microvesicles (100-350 nm), exosomes (30-150 nm), and apoptotic bodies (500-1000 nm), which are differentiated based upon their biogenesis, release pathways, size, content, and function. Exosomes can be formed by extracellular stimulation, microbial attack, and other stress conditions, and are released through either outward budding of the plasma membrane (microvesicle pathway) or inward budding of the endosomal membrane. Inward budding of endosomal membranes results in the progressive accumulation of intraluminal vesicles (ILVs) within the lumen of multivesicular endosomes (MVEs). MVEs fuse with the plasma membrane to release ILVs that are then called exosomes. The regulation of MVE and the formation and release of exosome are mostly mediated by the endosomal sorting complexes required for transport (ESCRT) machinery, or by the ESCRT-independent pathway.
Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).
Exosomes can be released by almost all kinds of cells under physiological and pathological conditions, and are widely distributed through body fluids. Exosomes play a key role in cell communication and epigenetic regulation by transporting proteins and genetic material. Analysis of exosome content to estimate the functional status of parental cells lays a foundation for exosome-based diagnosis. In addition to in disease diagnosis, exosomes are used in a variety of biomedical fields such as drug delivery, vaccine development, and regenerative medicine.
Exosome Detection by Flow Cytometry
Exosomes are difficult to identify because of their small size, strong heterogeneity, and low refractive index. The use of flow cytometry not only enables high-throughput analysis of exosomes, but also allows quantification or classification of exosomes based on their antigenic expression.
Exosomes are immobilized on the surface of the beads, and the exosomal vesicles are exposed to a fluorescently conjugated antibody against an antigen expressed on the exosome surface. When the sample passes through the laser of the flow cytometer, it emits a fluorescent signal which is detected.
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Exosome Detection Kits
Amerigo Scientific offers kits for isolation/detection of exosomes based on the immunobead assay method, which uses a bead-bound capture antibody and a fluorochrome conjugated detection antibody. The kits provide reproducible results and can be run in parallel to exosome immunophenotyping.
Allowing simultaneous immunophenotyping of exosomes capture population
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Figure 1. Exostep cytometric histogram showing the dynamic range (0-128µg) of exosomes from PC3 Cell line using ExoStep.
Figure 2. Sensitivity comparing among competitors.
Lyophilized Exosome Standards
Compared with fresh exosomes, lyophilized exosomes can be easily transported and stored at low temperature for a long time, and the complete morphology and biological function of exosomes can be maintained after solubilization. In addition to being used as a control standard for flow cytometry, lyophilized exosomes can also be used as a control standard for a variety of applications such as western blot (WB) and ELISA.
Amerigo Scientific offers the highest pure lyophilized exosome standards from human body fluids (plasma and serum) and different cell culture media. The lyophilized standards have been validated by WB and flow cytometry, for overall protein content and particle number by nanoparticles tracking analysis (NTA).
Highly pure exosomes, providing better performance than competitors
Guaranteed stability owing to an exclusive lyophilization procedure
Exhaustive validation by WB, NTA, flow cytometry and functional analysis in vitro
Tested in application for medicine regenerative, skin, dermatological and pharma companies
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Figure 3. Dynamic range of fresh (Left) and lyophilized (Right) PC3 exosomes analyzed by flow cytometry. Relationship between background noise and specific signal at different exosome concentrations. Exosomes were captured by CD63+ (Clone TEA3/18) capture beads and subsequently detected by Anti-CD9 PE (Clone VJ1/20).
Figure 4. Exosome analysis and comparative of fresh (Left) and lyophilized (Right) plasma exosomes for particle size and concentration by NTA, NanoSight LM10HSB. Analysis was carried out with 1 µl of purified exosomes diluted in 999 µl of HEPES buffer (dilution 1:1000). The purified exosomes showed a size distribution profiles, with peak diameters from 50 – 150 nm and concentrations about 1x1010 exosomes/ml.
Highly efficient methods for exosome isolation are prerequisites to obtain substantial breakthroughs. Amerigo Scientific offers common solutions for exosomes isolation from biological fluids, such as size exclusion chromatography, polymer precipitation, and immunoaffinity capture-based isolation.
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Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).
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