Exosome Services

The most commonly used are CD63, CD81, etc.

These protein markers were initially discovered through mass spectrometry analysis of purified extracellular vesicles as highly abundant proteins present in them. Therefore, they gradually began to be used as markers for extracellular vesicles. Actually, CD63, CD9, and CD81 are all members of the tetraspanin family. They are directly involved in the sorting of cargo into extracellular vesicles. TSG101 is a protein associated with the ESCRT complex, and ALIX is directly involved in the process of vesicle budding and scission from the membrane to form independent vesicular structures. These marker proteins are not unique to exosomes. It's primarily because they are mainly involved in the formation and secretion processes of vesicles; most are membrane proteins or ESCRT complex members and associated proteins. They originate in the cell and localize near intracellular membrane structures, so they are also present in cells. However, because the formation of extracellular vesicles depends on these molecules, and they function within vesicles, their abundance in vesicles is significantly higher than in cells.

In EM images, the morphology of exosomes should show a distinct cup-shaped or saucer-like structure. Generally, there is a slightly brighter rim at the edge of the exosome. If structures without a membrane are seen in EM results, they might not be exosomes; they could be lipoprotein particles or proteins.

NTA can be used for particle counting. However, quantifying exosomes based on particle number typically leads to overestimation because exosome samples may contain lipoproteins, protein aggregates, and other impurities that are also counted.

Theoretically, extracting RNA from tissue will also extract RNA from exosomes. However, the amount and variety of RNA extracted from tissue are certainly greater than those from exosomes. Because exosomes represent only a very small part relative to the tissue, and their contained materials are not equivalent.

Flow cytometry can detect exosomal marker proteins. Specific steps are as follows:

(1) Process the sample and extract exosomes.

(2) Antibody incubation: Resuspend the extracted exosomes in PBS containing 2% BSA. Perform primary and secondary antibody incubations sequentially to obtain the CD63-labeled exosome detection solution.

(3) Set flow cytometer parameters: Run the flow cytometer normally and set the parameters.

(4) Detection: Resuspend the CD63-labeled exosome detection solution in PBS and load it for detection.

(5) Use flow cytometry analysis software to analyze the data and obtain quantitative detection results for exosomes.

For most studies involving Exosome RNA isolation, we recommend using plasma. Serum is the liquid collected after blood coagulation, so it lacks fibrinogen and clotting factors, and contains many coagulation products. Fibrinogen converts to fibrin, which has clotting function. During serum collection and coagulation, platelets are stimulated and release many exosomes and other vesicles. Therefore, the number of vesicles obtained from serum is always higher than from plasma, with over 50% potentially derived from platelets. Thus, plasma is a better medium for studying exosomes in pathophysiological states. Therefore, plasma is generally chosen for experiments. However, when studying diseases related to platelets, serum should be prioritized.
During blood collection, cells and platelets should be removed by prompt centrifugation, usually within 30 minutes, avoiding long storage. Also, plasma (or serum) should be separated at room temperature. The centrifugation speed and rotor type should be consistent for all samples.

Membrane proteins are generally not recommended for boiling. Usually, after boiling the sample, the bands appear very smeared and cannot be recognized well by the antibody. This might be because some antibodies recognize conformational (3D) epitopes rather than linear epitopes.

Exosomes are extracellular nano-sized vesicles of endocytic origin. Circulating exosomes in body fluids can interact with recipient cells by carrying various functional molecules like lncRNA, miRNA, proteins, etc., mediating intercellular communication and influencing tumor occurrence and development. LncRNAs detected in exosomes are termed exosome-derived lncRNAs. Studies have found that exosomes can transfer tumor-associated lncRNAs to tissue cells, affecting tumor biological processes, playing important roles in tumor proliferation, metastasis, invasion, drug resistance, immune regulation, and angiogenesis. Furthermore, tumor-derived exosomes have the ability to precondition distant or specific sites. Transferring lncRNAs to recipient cells at these sites can create a pre-metastatic niche suitable for tumor cell growth. Therefore, changes in the tumor microenvironment may lead to altered lncRNA content within the tumor tissue itself.

For loading microRNA into exosomes, consider incubation, sonication, extrusion, freeze-thaw, or chemical transfection methods. These methods can allow drugs to enter the interior of exosomes easily. After drug diffusion, the exosome membrane reseals.

The incubation method is relatively simple, does not disrupt exosome membrane integrity, and is suitable for loading hydrophobic small molecules. Electroporation is mainly suitable for loading hydrophilic molecules with high efficiency but may compromise exosome membrane stability. If considering cost and loading efficiency, the incubation method can be chosen.

Besides these, the following methods can also be used to load microRNA:

1.Sonication: During sonication, using an ultrasonic homogenizer probe causes membrane deformation, allowing drug diffusion into exosomes.

2.Extrusion: The exosome-drug mixture is passed through a membrane with pores of 100-400 nm using a lipid extruder to transfer the drug into the exosomes.

3.Freeze-thaw: The exosome-drug mixture is frozen repeatedly (at -80°C or in liquid nitrogen) for several cycles and then thawed to room temperature to ensure successful drug incorporation.

4.Chemical transfection: Exosomes and drugs are incubated with surfactants, causing pore formation in the membrane, allowing drug permeation. The most commonly used surfactant is saponin, hence this method is also called saponin-assisted loading.