The 2013 Nobel Prize in Physiology or Medicine was jointly awarded to three scientists for their discovery of vesicle transport in cells, a clear indication of the importance of vesicles in physiology and their applied potential in diagnostics and therapy.
Exosomes are now believed to be present in all body fluids, and represent a new way of thinking about cell signaling. These small (30-150nm) extracellular vesicles are thought to play a role in a large number of biological functions.
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A range of specific tools and technologies are needed for the isolation and analysis of exosomes and their cargo. The products presented here have been developed to simplify the overall study of exosomes, their composition, and function.
Ultracentrifugation can be tedious and time consuming. Now you can easily enrich for intact exosomes from a variety of starting samples using the flexible and scalable Total Exosome Isolation reagents. These reagents and accompanying protocols are ideal for a range of experimental set-ups including low volume input and handling of multiple samples.
Total exosomes enriched from cell culture (by using the Total Exosome Isolation reagents or ultracentrifugation) can be further purified into specific subpopulations by immunomagnetic capture. Use the Dynabeads®-based CD9, CD63, CD81, or EpCam-specific reagent, or combine the Streptavidin reagent with your choice of a biotinylated antibody to purify any population based on a surface antigen.
|- Molecular assays (e.g. PCR, Western)
- Biological assays (e.g. tracing exosomes in vivo)
- Intact exosome analysis (e.g. flow and EM)
|- Molecular assays
- Intact exosome analysis
Visual identification of exosomal vesicles can be challenging, and free exosomes alone are too small to be detected in flow. There are a few methods, though, which can be used for analysis.
Exosomes recovered with the Total Exosome Isolation reagent can be analyzed using the NanoSight® instrument for approximate size range and concentration.
Exosomal RNA or membrane components can be labeled, allowing for visualization of exosomes under the microscope. Specialized spin columns are available to remove unincorporated dyes. These columns also allow for buffer exchange and easier desalting or removal of low molecular weight (< MW 3000) contaminants compared to current methods.
Electron Microscopy (EM)
One of the advantages of using Dynabeads® magnetic separation technology to pull out pure and specific exosomes is that you can move directly to EM analysis. You can analyze subsets from total exosomes enriched from cell culture using CD9, CD63, CD81, or EpCam-specific reagent, or use the Streptavidin reagent in combination with your choice of biotinylated antibody.
Immunomagnetic capture of exosomes on the surface of the Dynabeads® allow for a clear and defined FFC/SSC detection typically in less than one hour. You can analyze subsets from total exosomes enriched from cell culture using the CD63-specific reagent, or use the Streptavidin reagent in combination with your choice of biotinylated antibody.
Exosomes have been shown to transport a range of molecules from one cell to another. Their cargo includes proteins, lipids, mRNA (fragments and full length), rRNA, miRNA, and various ncRNA.
RNA & Protein Isolation
Simultaneous isolation of both proteins and total RNA from the same sample of pre-enriched exosomes can be achieved using the Total Exosome RNA and Protein Isolation Kit. The kit is compatible with all protocols for exosome isolation.
Specific monoclonal antibodies (CD9, CD63 and CD81) allow for detection of cellular and exosomal antigens by Western blot analysis. If you are looking to compare multiple protein samples on the same gel and want to keep exosomal protein complexes intact, you should check out the fast and gentle Dynabeads®-based immunoprecipitation method using Protein A or Protein G in combination with your primary antibody of choice.
Exosomal RNA recovered using the Total Exosome RNA and Protein Isolation Kit can be analyzed e.g. by qRT-PCR using TaqMan® assays, RNA sequencing, or next generation sequencing tools. Exosomal RNA and membrane components can be labeled using fluorescent dyes, while unincorporated dye can be removed by using dedicated spin columns.
We asked 10 prominent scientists to share their thoughts on exosomes research.
Don’t miss any of the episodes:
ISEV 2013 Presentation:
"RNA profiling of exosomes"
ISEV 2013 Presentation:
"From isolation to characterization of exosomes"
Isolation and characterization of RNA content of human blood-derived exosomes
These published articles are all citing the use of exosome-specific products from Life Technologies:
- Expression of B-Cell Surface Antigens in Subpopulations of Exosomes Released From B-Cell Lymphoma Cells
Morten P. Oksvold,PhD; Anette Kullmann,BS; Lise Forfang,BS; Bente Kierulf,BS; Mu Li,PhD; Andreas Brech,PhD; Alexander V. Vlassov,PhD; Erlend B.Smeland,MD,PhD; Axl Neurauter,MSc; and Ketil W. Pedersen,PhD.
- MicroRNA-193b is a regulator of amyloid precursor protein in the blood and cerebrospinal fluid derived exosomal microRNA-193b is a biomarker of Alzheimer's disease
Liu CG et.al. View abstract
- Hepatocellular Expression of B-Cell Surface Antigens in Subpopulations of Exosomes Released From B-Cell Lymphoma Cells
Oksvold MP et. al. View Abstract
- Expression of Serum Exosomal MicroRNA-21 in Human Hepatocellular Carcinoma
Wang H et. al. View Abstract
- Programmed Cell Death 4 and MicroRNA 21 Inverse Expression Is Maintained in Cells and Exosomes From Ovarian Serous Carcinoma Effusions
Cappellesso R et. al. View Abstract
- Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs
Chen W-x et. al. View Abstract
- miR-145 suppresses thyroid cancer growth and metastasis and targets AKT3
Boufraqech M et. al. View Abstract
- Natural Killer Cell-Mediated Shedding of ULBP2
Wang R et. al. View Abstract
- Histone Deacetylase 3 Unconventional Splicing Mediates Endothelial-to-mesenchymal Transition through Transforming Growth Factor β2
Zeng L et.al. View Abstract
- Methods for the extraction and RNA profiling of exosomes
Zeringer E et. al. View Abstract
- Delivery of Functional Anti-miR-9 by Mesenchymal Stem Cell–derived Exosomes to Glioblastoma Multiforme Cells Conferred Chemosensitivity
Munoz JL et.al. View Abstract
- The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo
Schageman J et. al. View Abstract
- Characterization of a Stem-like Subpopulation in Basal-like Ductal Carcinoma in Situ (DCIS) Lesions
Li Q et.al. View Abstract
- Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials
Vlassov AV et. al. View Abstract
- Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells
Thayanithya V et.al. View Abstract
- Systemically Circulating Viral and Tumor-Derived MicroRNAs in KSHV-Associated Malignancies
Chugh PE et.al. View Abstract
- Differences in exosome populations in human breast milk in relation to allergic sensitization and lifestyle
Torregrosa Paredes P et.al. View Abstract
- Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers
Li M et. al. View Abstract
- Labeling exosomal components using fluorescent dyes