Exosome Products for Isolation, Detection, and Analysis

Exosomes are small (30–150 nm) vesicles containing DNA, RNA, proteins, lipids, and metabolites. Exosomes are constantly secreted by cells in vitro and in vivo. The precise molecular mechanics for their secretion and uptake as well as the composition of their cargo, and resulting functions, are still being discovered. Exosomes have become the focus of growing interest both to study their functions and to understand ways to potentially use them as treatments for diseases.

Exosomes’ primary function is to mediate near and long-distance intracellular communication, by transporting their cargo of nucleic acids, proteins, lipids, amino acids, and metabolites, reflecting their cell of origin. Exosomes are being investigated for the key roles they play in the following processes:

• Intracellular communication
• Facilitation of the immune response
• Apoptosis
• Angiogenesis
• Inflammation
• Metastasis and progression of tumors
• Blood coagulation

Exosome isolation using ultracentrifugation is considered a traditional method of isolation that can be both time-consuming and can damage the exosomes, changing their morphology and functional properties. Exosomes can be separated from cells and heavy artifacts by tying up water molecules via polymer precipitation, thereby reducing the solubility of exosomes and bringing about their precipitation. Exosomes are then harvested following low-speed centrifugation. Total Exosome Isolation reagents and kits utilize this method and help speed up the process of exosome isolation beginning with sample collection—from cell culture media, serum, plasma, urine, or other body fluids—and ending with purified exosomes ready for downstream molecular analyses (Figure 1). The Total Exosome Isolation kits and reagents, and accompanying protocols, are ideal for a wide range of experiments, including processing low sample volumes and handling multiple samples.

Figure 1. Traditional method of exosome isolation via ultracentrifugation.

Exosome isolation using immunomagnetic separation is a method particularly suited to purify exosome subpopulations. Exosome-specific antibodies are bound to superparamagnetic beads for more efficient purification of exosomes. Exosome-Streptavidin Isolation/Detection Reagent helps isolate and detect exosomes when combined with a biotinylated primary antibody. These exosomes can then be detected using assays such as flow cytometry, electron microscopy, and western blot (Figure 2).

Figure 2. Another method of exosome isolation via immunomagnetic separation.

Antibody-specific exosome isolation reagents are also available to isolate exosome subpopulations. These antibodies include:

• CD9
• CD63
• CD81
• EPCAM

Following isolation, the specific subpopulation of exosomes can be further studied using methods such as western blot, qPCR, or next-generation sequencing.

View all exosome isolation products

Detection and analysis of exosomes can be very challenging due to their small size (30–150 nm). However, there are a few methods used for exosome detection and analysis.

• NanoSight instrument—Exosomes recovered with Total Exosome Isolation reagents and kits can be sized and counted using a NanoSight instrument or similar tool, such as ParticleMetrix, Izon, and Malvern.
• Light microscopy—Exosomal RNA or membrane components can be labeled, allowing for visualization of exosomes under the microscope.
• Electron microscopy (EM)—One of the advantages of using Dynabeads magnetic beads to purify exosomal subpopulations is that you can directly perform EM analysis. You can analyze subsets from total exosomes enriched from cell culture using CD9-, CD63-, CD81-, or EPCAM-specific reagents, or have the option to use the Streptavidin reagent in combination with your choice of biotinylated antibody.
• Flow cytometry—Immunomagnetic capture of exosomes via Dynabeads magnetic beads help allow for clear and defined FFC/SSC detection (typically in less than one hour). You can analyze subset exosome populations using the CD9-, CD81-, or EPCAM-specific reagents, or have the option to use the Streptavidin reagent in combination with your choice of biotinylated antibody.

Exosome subpopulations can be defined by their surface receptor. Antibodies specific to these surface receptors are used to detect exosome subpopulations. The following antibody-specific exosome analysis reagents for exosome subpopulations include:

• CD9
• CD63
• CD81
• EPCAM

Following isolation, the specific subpopulation of exosomes can be further studied using methods such as western blot, qPCR, or next-generation sequencing.

View all exosome detection and analysis products

Exosomes have been shown to transport a range of molecules between different cell types. Their cargo includes various proteins, mRNA (fragments and full length), rRNA, tRNA, miRNA, and ncRNA.

• RNA isolation—Purification of total RNA, or recovery of both protein and RNA from the same sample of pre-enriched exosomes, can be achieved using the Invitrogen Total Exosome RNA and Protein Isolation Kit. The kit is compatible with all protocols for exosome isolation.
• Protein isolation—Exosome Immunoprecipitation Reagents (Protein A or Protein G) are designed for the immunoprecipitation of proteins, protein complexes, protein-nucleic acid complexes, and other exosome-related antigens, which can then be used in molecular assays and intact exosome analysis.
• Protein analysis—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 may use the Dynabeads-based immunoprecipitation method using Protein A or Protein G.
• RNA analysis—Exosomal RNA and membrane components can be labeled using fluorescent dyes, while unincorporated dye can be easily depleted with Invitrogen spin columns. Exosomal RNA recovered using the Total Exosome RNA and Protein Isolation Kit can be analyzed by qRT-PCR using Applied Biosystems 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 via spin columns.