Beckman Coulter

Isolating Exosomes, Steps Towards Standardization

An improved and more efficient isolation and characterization protocol for exosomes and other extracellular vesicles (EVs) is critical to advancing this exciting field, and experts have recently called for the establishment of standardized methods. Ultracentrifugation is the gold standard1 for the isolation and purification of exosomes from biological fluids and cell culture samples, and enables accurate and reproducible yield of exosomes, while minimizing co-purification of protein aggregates and other membranous particles. Downstream challenges involve a standardized method for genetic profiling of encapsulated miRNA. The following white papers offer a look at using Biomek methods for centrifugation layering and fractionation, total RNA extraction, and cDNA amplification and clean-up for next generation sequencing. Recent studies have suggested that exosomes can serve as biomarkers for future clinical and diagnostic use in not only cancer, but many other human diseases2. Exciting new findings have implicated exosomes in cardiovascular diseases3-5, autoimmune syndromes6, and neurodegenerative disorders such as Alzheimer’s7 and Parkinson’s8 disease, in addition to infectious diseases such as tuberculosis9, diphtheria10, and even HIV11, and standardization of processes is an essential step for reliable and interpretable results. 

 

A Standardized, Automated Approach for Exosome Isolation and Characterization Using Beckman Coulter Instrumentation

This white paper details the protocol for exosome isolation and sequencing of exosome-derived RNA.  

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Exosome-Depleted FBS Using Beckman Coulter Centrifugation

This white paper details the importance of exosome depletion of media and additives for use in exosome isolation studies.

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g-Max: Added Capabilities to Beckman Coulter’s Versatile Ultracentrifuge Line

This white paper details Beckman Coulter’s g-Max System, and how it can save you precious time during your exosome isolation procedure.

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References:

  1. Momen-Heravi, F., Balaj, L., Alian, S., Mantel, P-Y., Halleck, A.E., Trachtenberg, A.J., et. al. Current methods for the isolation of extracellular vesicles. Biol. Chem. 2013. Doi: 10.1515/hsz-2013-0141.
  2. De Toro, J., Herschlik, L., Waldner, C., Mongini, C.. Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Front. Immunol. 2015. Doi: 10.3389/fimmu.2015.00203.
  3. Amabile, N., Rautou, P-E, Tedgui, A., Boulanger, C.M.. Microparticles: key protagonists in cardiovascular disorders. Semin Thromb. Hemost. (2010) 36:907–16. doi:10.1055/s-0030-1267044Asdf.
  4. DeJong O.G., Verhaar, M.C., Chen, Y., Vader, P., Gremmels, H., Posthuma, G., et. al. Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes. J Extracell. Vesicles (2012) 1:18396. doi:10.3402/jev.v1i0.18396.
  5. Waldenström, A., Gennebäck, N., Hellman, U., Ronquist, G.. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells. PLoS One (2012) 7:e34653. doi:10.1371/journal.pone.0034653.
  6. Robbins, P.D., Morelli, A.E.. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol (2014) 14:195–208. doi:10.1038/nri3622.
  7. Rajendran, L., Honsho,M., Zahn,T.R., Keller,P., Geiger,K.D., Verkade,P., et. al. Alzheimer’s disease beta-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. USA (2006) 103:11172–7. Doi:10.1073/pnas.0603838103.
  8. Danzer, K.M., Kranich, L.R., Ruf, W.P., Cagsal-Getkin, O., Winslow, A.R., Zhu,L., et. al. Exosomal cell-to-cell transmission of alphasynuclein oligomers. Mol Neurodegener (2012) 7:42. doi:10.1186/1750-1326-7-42.
  9. Kruh-Garcia, N.A., Wolfe, L.M., Chaisson, L.H.,Worodria, W.O., Nahid P., Schorey J.S., et. al. Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS. PLoS One (2014) 9:e103811. doi:10.1371/journal.pone.0103811.
  10. Colino, J., Snapper, C.M. Exosomes from bone marrow dendritic cells pulsed with diphtheria toxoid preferentially induce type1 antigen-specific IgG responses in naïve recipients in the absence of free antigen. J Immunol (2006) 177:3757–62. doi:10.4049/jimmunol.177.6.3757.
  11. Gould S.J., Booth, A.M., Hildreth,J.E.K.. The Trojan exosome hypothesis. Proc Natl Acad Sci USA (2003) 100:10592–7. doi:10.1073/pnas.1831413100.

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