Exosome Products for Isolation, Detection, and Analysis

Exosomes are a subset of small (30–150 nm) extracellular 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
• Pathological processes in cardiovascular diseases, neurodegeneration, and cancer

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. Isolation of exosomes using precipitation and low-speed centrifugation.

Dynabeads magnetic beads and DynaGreen magnetic beads are essential tools for isolating and detecting exosomes in various research applications. Magnetic beads offer a simple isolation process from different starting materials such as cell culture media or liquid biopsy samples. Additionally, the KingFisher Sample Purification Systems can automate the process for high throughput or screening.

The following products include magnetic beads pre-conjugated with antibodies for isolation of specific exosome sub-populations: Exosome-Human CD63 Isolation/Detection Reagent, Exosome-Human CD81 Isolation Reagent, Exosome-Human CD9 Isolation Reagent, and Exosome-Human EpCAM Isolation Reagent. These beads isolate exosomes from cell culture media during an overnight incubation.

To enhance flexibility, biotinylated antibodies can be combined with the Exosome-Streptavidin Isolation/Detection Reagent to isolate specific sub-populations using various exosome-specific markers. Alternatively, IgG antibodies from any species can be combined with our sustainable DynaGreen CaptureSelect Anti-IgG-Fc (Multi-Species) Magnetic Beads to capture the exosome of choice. This isolation process is fast, automatable, and efficient, and takes only 40 minutes using liquid biopsy samples such as plasma and urine. Once the exosomes are isolated, they can be further analyzed with western blot, mass spectrometry, qPCR, or electron microscopy (Figure 2).

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Exosome isolation from liquid biopsy samples refers to the process of separating exosomes from various body fluids, such as serum (Figure 3), plasma, and urine. The isolation process involves separating exosomes from other components in the liquid biopsy sample, such as cells, proteins, and debris, to obtain a pure exosome population. Dynabeads Intact Virus Enrichment isolates exosomes from liquid biopsy samples in less than 15 minutes and enables automation for a high-throughput enrichment. The enrichment is charge-based, thus the negatively charged exosomes in the sample bind to the positively charged Dynabeads magnetic beads. Changing the buffer conditions allows for an easy and quick release step.

The product can isolate both negatively charged viruses and exosomes.

Figure 3. Reversible exosome enrichment from serum. SW480 exosomes were spiked into serum followed by isolation with the Dynabeads Intact Virus Enrichment beads. The exosomes were released from the magnetic beads with 0.25M KI. The serum used was undiluted or diluted to 10% and 50% in PBS. Released exosomes were analyzed by Western blot detecting CD81 (A) or by re-capture by Exosome-Human CD81 Flow Detection Reagent for downstream flow cytometry (B-G). Exosomes spiked into 10%, 50% and 100% serum were captured and released from the Dynabeads Intact Virus Enrichment beads and detected by Western blot (A) and flow cytometry (B-E). CD81 was not detected on the Dynabeads Intact Virus Enrichment beads after release, suggesting that most of the exosomes were eluted off the beads. Exosomes spiked into PBS or 10% serum demonstrated similar isolation and release efficiency (C and D) and in the same range as prior to isolation (B). Interestingly, less dilute serum samples (50% and 100%) resulted in slightly lower capture efficiency (E and F). Exosomes captured from un-spiked and undiluted serum samples were included, demonstrating that a small fraction of the captured exosomes are native exosomes present in the sample (G). Each sample is compared to the isotype control using an irrelevant staining antibody of the same isotype (gray histograms).

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.

• Nanoparticle tracking 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.
• 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 Exosome-Human CD9 Isolation Reagent-, Exosome-Human CD63 Isolation/Detection Reagent-, Exosome-Human CD81 Isolation Reagent-, or Exosome-Human EpCAM Isolation Reagent, 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 Exosome-Human CD63 Isolation/Detection Reagent, Exosome-Human CD9 Flow Detection Reagent, Exosome-Human CD81 Flow Detection Reagent, or Exosome-Human EpCAM Flow Detection Reagent
• Dynamic light scattering
• HS flow cytometry
• Resistive pulse sensing (Spectradyne)

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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 (Exosome Anti-Human CD9, Exosome Anti-Human CD63, and Exosome Anti-Human 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.