For your convenience, Creative Biolabs has grouped these questions & answers into the following categories.
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Q: What are exosomes?
A: Exosomes are a subtype of extracellular vesicles (30-150 nm) that are responsible for cell to cell communication. Exosomes carry proteins and RNAs, which are released naturally by exocytosis into the extracellular space. They are secreted by most cell types and are found in all bodily fluids such as plasma, urine, serum, saliva and cerebrospinal fluid (CSF).
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Q: What is the composition of exosomes?
A: Exosomes are microvesicles composed of a lipid bilayer and a liquid cargo. Exosomes may contain cytosols, biologically active molecules, and other components from cells of their origin, including lipid rafts and membrane surface proteins.
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Q: What are the similarities and differences between exosomes and microvesicles?
A: Both exosomes and microvesicles are membrane-bound vesicles that can deliver functional mRNA, miRNA, and proteins to receptor cells. The differences between the two types of vesicles are based on their biogenesis and biophysical properties, including size and surface protein markers.
Exosomes are smaller in diameter than microvesicles (30-150 nm vs 100-1000 nm). Exosome biogenesis, the release of protein cargo classification involves the transport of the required endosomal classification complex (ESCRT complex) and other related proteins, such as CD63, CD9, Alix and TSG101. In contrast, microvesicles are produced directly through the outward budding and division of membrane vesicles. Therefore, their surface markers largely depend on the composition of the original membrane.
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Q: Why exosomes get so much attention?
A: First, exosomes are thought to be mediators for cellular communication and intercellular macromolecular transport.
Second, exosomes are thought to play an important role in the transmission of proteins, lipids, mRNA, miRNA, and DNA that have been linked to normal and pathological processes. They could become the cause of many diseases.
Finally, exosomes have a lipid bilayer membrane structure that allows them to develop into natural carriers for specific drugs. They can protect the coatings and target specific cells or tissues.
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Q: What are the functions of exosomes?
A: During normal development and homeostasis, exosomes play an important role in intracellular communication, enabling bioactive components (DNA, RNA and proteins) to be transferred between cells. Exosomes are also linked to disease states, including cancer, infectious diseases, and neurodegenerative diseases. Due to the high availability of exosomes in body fluids, exosomes are being studied as the markers of disease and as a way to understand disease progression.
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Q: What approaches does Creative Biolabs utilize in exosome isolation?
A: Creative Biolabs has developed various methods for exosome isolation from a wide of sources. These methods include ultracentrifugation, polymer-based precipitation, size exclusion chromatography and affinity-based capture. We utilize these methods to assist you in isolating high-yield and high-purity exosomes from a variety of sources, including almost any biofluid (such as plasma, urine, serum, CSF, ascites fluid, and saliva) and multiple cell types (such as tumor cells and B cells).
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Q: Is there a risk that my exosomes will be damaged during isolation by ultracentrifugation?
A: Ultracentrifugation is a robust and reliable way for exosome isolation and purification. However, some degrees of sample loss can occur due to apoptosis and incomplete sedimentation. According to our experience, fewer steps involved in the isolation can yield better results. Ultracentrifugation is a pre-enrichment step that will inevitably cause loss of vesicles even with caution. Care should be taken to avoid steps such as vigorous agitation, detergents, and freezing, which could lead to exosome lysing.
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Q: When do I need to isolate specific exosomes?
A: If you are interested in exosomes expressing selective and specific exosomal markers, specific isolation method is needed. In this case, we recommend immunocapture methods, such as using immunobeads or immunoplates.
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Q: How do you get specific exosome sub-populations?
A: Our scientists are proficient in isolating any exosome subtype by targeting surface markers. We have developed an efficient strategy to classify and characterize exosomes.
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Q: Do exosome isolation reagents precipitate apoptotic bodies?
A: No. Apoptotic bodies are >800 nm, which are much larger than exosomes (30-150 nm), so they can be mostly removed along with cells and debris during the pre-spin step. The exosome reagents are added in the following step to precipitates primarily exosomes.
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Q: Can you isolate exosomes from bacteria or fungus or parasite samples? How to distinguish them?
A: Yes, we have used conventional ultracentrifugation methods to isolate exosomes from bacteria, fungus, and parasite samples.
If you need to distinguish exosomes from different sources, it is best to separate the samples before isolating the exosomes. If it is difficult to separate the samples when they are cultured together, you can choose the corresponding database for specific analysis when doing NGS and MS detection.
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Q: Can exosomes be recovered from viral particles? If so, how?
A: It is very difficult to isolate exosomes from viral particles due to the similarity in size and buoyancy density between exosomes and viruses.
It has been found that exosomes migrate more slowly in density gradient than viral particles to get separated from the corresponding viruses. Neutralizing antibodies can also be used to distinguish exosomes from non-enveloped viruses. In addition, a high-throughput method for the analysis of single nano-scale particles may help to distinguish different particles in the exosome-virus continuum. This flow cytometry based separation technology based not only includes the hardware transformation in high-end flow cytometer to improve the signal-to-noise ratio, but also includes the optimized staining scheme for universal labeling of exosomes, as well as the use of magnetic beads to screen the surface antigen components of exosomes.
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Q: Does cell death affect the study of exosome?
A: Yes, apoptotic cells release a lot of apoptotic vesicles and cell death absolutely affects exosomes secretion. Therefore, we always recommend using healthy and viable cells for the exosome study.
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Q: Can exosomes be secreted by quiescent cells?
A: During the recycling of the plasma membrane or intercellular communication, exosomes can be formed. Hence, we can say that exosomes can also be secreted by quiescent cells.
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Q: Can I use DMSO in serum sample for studying exosomes?
A: DMSO is a cryoprotectant usually used in storage buffer to protect the cell membrane from any damage that may be caused by freezing. However, compared to the cells, the size of the exosomes is very small with little liquid content. Thus, we do not recommend using DMSO while freezing your serum samples.
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Q: When I need to isolate exosome from plasma, can I use heparin or EDTA tube to collect blood sample?
A: No. Because heparin will significantly affect the downstream RNA analysis and EDTA may interfere with the downstream PCR assay.
Use a No-heparin-No-EDTA tube to collect the blood sample. If anticoagulant has to be used, use EDTA tube to collect the blood and adjust the concentration of Mg2+ in the PCR reaction.
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Q: What's sample requirement for exosome isolation? How much starting material do I need for exosome isolation?
A: Samples can be any type of biofluid or cell culture media that contains exosomes. The starting volumes of available isolation protocols is >100 μL. There is no upper limit for large sample volumes. The amount of starting material depends on the fluid type, the isolation method and the downstream analyses. The concentration of exosomes varies across fluid types and disease states. A small volume of starting material can be used first to estimate the yields before processing a larger volume.
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Q: If the amount of serum / plasma is small, how to isolate exosomes?
A: In general, ultracentrifugation is a standard method for the isolation of exosomes. However, this method is not suitable for samples of small volume. Instead, an exosome isolation kit is recommended. The minimum volume to be used with the kit is 100 μL. However, since the quantity of exosomes could vary between samples, we always recommend starting with larger amount of sample, especially for urine and cell supernatant samples, for which we recommend the starting sample volume to be at least 1mL.
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Q: Can you provide a full range of MSC-derived exosome extraction?
A: Yes, Creative Biolabs can provide a one-stop service that begins from cell culture to exosomes isolation. In addition, in order to meet the needs of large-scale preparation, the isolated exosomes can be powdered by freeze-drying technology for convenient for transportation and long-term storage.
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Q: Can you provide GMP level exosomes?
A: We have established a large-scale production line for exosomes separation. For cell culture supernatant samples, the maximum daily processing capacity can reach 10 L. At present, we are still actively establishing a GMP-level production line, which will be put into use soon.
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Q: Can you recommend a spin concentrator to concentrate the cell media samples from 10 ml to 1 ml.
A: Spin concentrators with 3000 and 10,000 cutoff are available from any companies.
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What applications does your isolation platform support?
A: There are almost no limits to downstream applications. We have helped researchers isolate exosomes for use in downstream analyses involving DNA, RNA, and proteins, ranging from diagnostics to therapeutics.
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Q: How to distinguish exosomes from other types of vesicles?
A: According to the proposal of the ISEV in 2018, EVs characterization should include:
1) at least three positive protein markers of EVs should be identified;
2) nanoparticle tracking analysis (NTA);
3) transmission electron microscope (TEM) for EV morphological characterization.
Specifically, in addition to classifying 30-150 nm of the vesicles as exosomes, the vesicles should also be positive for certain protein surface markers, such as tetraspanins (CD63, CD81, CD9) and others like ALIX. Western blotting of these targets on interested samples is a relatively simple method to confirm that vesicles are indeed exosomes. At the same time, the samples also need to be analyzed by electron microscopy, and the morphology of exosomes is disk like.
In Creative Biolabs, we have several WB marker kits to verify the presence of membrane in exosomes:
Exosomal Marker CD63 Antibody Set Kit
Exosomal Marker CD81 Antibody Set Kit
Exosomal Marker Alix Antibody Set Kit
Exosomal Marker Tsg101 Antibody Set Kit
Exosomal Marker CD9 Antibody Set Kit
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Q: Are factors such as CD9 or CD81 specific for exosome origin?
A: At present, there is no agreement on general exosome markers. The current proposal from the research community is to jointly detect many membrane-bound or membrane-associated proteins to verify the existence of the membrane. Our experience is that targeted CD63 and CD81 have been found in many different exosomes, as has CD9. However, some research has shown CD9 to be negative in exosomes released by at least two different cell lines (Jurkat cells and several B-cell lymphoma cells, Oksvold, 2014).
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Q: What are the biomarkers used to identify / characterize the isolated exosomes?
A: According to the literature reports and our experience, the markers that can be used as exosomes are common tetraspanins (CD9, CD63, and CD81 proteins), heat shock proteins (HSC70 and HSC90), membrane transporters (GTPases) and ESCRT proteins. Moreover, TSG101, Adam10, ALIX, flotillin are also indicated for detecting exosomes isolated from cell media and human biofluids.
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Q: How to prepare exosome samples for Western blot analysis?
A: We recommend using non-reducing samples for exosome proteins. There should not be DTT or beta-mercaptoethanol during sample boiling before loading onto the PAGE gel.
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Q: What are the negative controls for exosome isolation?
A: Several negative controls can be used, including but not limited to Grp94HSP90B1, endoplasmic reticulum, calnexin (CANX), Golgi (GM130), nucleus (histonesHIST*H*); Argonaute/RISC complex (AGO*) and mitochondria (cytochrome CCYC1). These proteins should be absent from exosomes.
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Q: How to distinguish apoptotic bodies and exosomes from the same cell?
A: Annexin V, thrombospondin and C3b are typical markers of apoptotic bodies and can be used to distinguish them from exosomes.
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Q: For exosomes isolated from adipocytes, is there any specific marker?
A: Studies have shown that almost all adipocyte-derived exosomes express fatty acid binding protein (FABP4). Therefore, FABP4 can be a good marker for exosomes isolated from adipocytes.
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Q: Is there any specific marker for apoptotic bodies and exosomes originated from same cells?
A: C3b, annexin V and thrombospondin are typical markers of apoptotic bodies and can be used to differentiate them from exosomes.
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Q: How do I visualize microvesicles, apoptotic bodies and exosome pellet from centrifugation?
A: An exosomes pellet cannot be distinguished from an apoptotic bodies pellet after centrifugation. You can use flow cytometry (FACS), WB, mass spectrometry (MS), electron microscopy and atomic force microscopy to characterize individual extracellular vesicles. Cytospins and Immunofluorescent images can be used for the identification of larger vesicles including apoptotic bodies. Nanosight Analysis and Dynamic Light Scattering can be used to identify size distribution.
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Q: What are the methods for exosome quantification?
A: Creative Biolabs has successfully launched an innovative exosome quantification platform with multiple high-end technologies (including nanoparticle tracking analysis, tunable resistive pulse sensing, ELISA, vesicle flow cytometry and surface plasmon resonance) to detect and quantify exosomes in complex biological samples. See our exosome quantification service for more details.
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Q: How do you define yield and purity? What is the exosome yield per cell?
A: Yield can be determined by using our standard exosome solutions. We use their known values as a basis for input in order to measure the output of own solutions. Purity can be evaluated by nano tracking analysis (NTA) based on size distribution (in which exosomes are defined as vesicles smaller than 200 nm), surface marker immunostaining, or transmission electron microscopy (TEM) to visualize the exosomes.
Exosome yield per cell depends on several factors, such as cell type, the culture conditions, the uniformity in the size of exosomes, and the time of harvest. We cannot give an exact exosome yield per cell because it cannot be a fixed number.
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Q: In an exosome study, can the content of the corresponding exosome be determined based on the protein concentration?
A: The concentration of exosomes within a solution is usually assessed by protein analysis (either Western blot or Bradford analysis). Western blot analysis of the protein content of exosomes is mainly aimed at the antibodies against one of the main proteins (such as CD63) commonly found in exosomes. Because the size and composition of the exosomes are different, this value cannot be extrapolated directly to the number of exosomes. But for most applications, assessing the relative amount of exosomal protein is sufficient to indicate the proportion of exosomes.
However, certain applications require information about the absolute number of exosomes present in the solution, such as the use of exosomes to deliver therapeutic agents. Quantification will be an important step in standardizing the number and size of exosomes used as cargo delivery particles. There are three possible methods for direct evaluation of exosomes: electron microscopy, nanoparticle tracking analysis, ELISA based quantitative immunoassay or a combination of these techniques.
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Q: How much protein concentration of exosomes is appropriate for WB?
A: After the exosomes are dissolved, we usually use Bradford or BCA method to determine the protein concentration in vesicles. But in WB detection, we found that the protein content was different from the number of exosomes. For accuracy, we give the minimum amount of samples from several different sources:
Exosomes: > 100 μl
Plasma / Serum: > 2 ml
Cell culture supernatant: > 10 ml
Urine: > 40 ml
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Q: Can the purified exosomes after isolation be frozen? Does the freezing step affect the exosome structure? What is the best way to store exosomes?
A: Since fresh samples are always better than frozen ones, exosomes should be processed freshly. However, samples in -80°C that were frozen immediately after isolation and were not freeze thawed multiple times, can also be used. The exosomal membrane is rich in phospholipids, sphingolipid, ceramides and tetraspanin proteins such as CD63, CD81 and CD9 that enforce the stability of the membrane. However, the freeze-thaw cycles might affect the morphology. For storage, we keep exosomes in PBS with 0.1% BSA, because this buffer is also used to dissolve the pellet after precipitation. For short-term, exosomes can be stored at 4°C for up to 1 week. For long-term storage, exosomes can be stored at -20°C or -80°C. We suggest aliquoting the exosome suspensions into multiple tubes to avoid freezing/thawing repeatedly.
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Q: Can the exosomes be frozen/thawed repeatedly?
A: We do not recommend repeatedly freezing/thawing exosome samples because this may induce an increase in particle concentration and in the width of the size distribution. We suggest aliquoting your exosomes after the initial thawing.
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Q: What is the best storage condition of cell media for subsequent isolation of exosomes?
A: Fresh samples are the best, but cell media can be stored at 4°C for up to one week. For long term storage, -80°C is recommended.
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Q: What is the storage condition for biological fluids before exosome isolation?
A: Most biological fluids (urine, saliva, and semen,) can be stored at -80°C before exosome isolation.
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Q: Are the storage conditions of exosomes isolated from different sources the same?
A: We have not seen any difference on the effect of storage conditions between exosome sources. Store purified exosomes at -20°C for frequent use and at -80 °C for long term storage.
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Q: As a carrier, what cargoes can exosomes carry?
A: As a medium of intercellular communication, exosomes can be used as carriers. They can transport bioactive molecules between cells, including proteins, lipids, DNA, RNA, microRNA, mRNA and lncRNA.
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Q: Why are exosomal miRNAs so significant in cancer diagnosis and treatment?
A: The intercommunication of cancer cells with their surrounding and distant environments is the key to tumor survival, progression, and metastasis. Exosomes are intercellular communication tools between cancer cells and other cells in the microenvironment. The miRNA expression in cancer cells is frequently dysregulated and can be reflected by the unique exosome miRNA isolated from the body fluids of cancer patients. Therefore, exosomes miRNAs are expected to be the promising tumor therapeutic agents in the future.
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Q: What genes can be used to normalize the expression of mRNA in exosomes?
A: There are many miRNAs that can be used for normalization, such as miRNA16, miRNA26a, miRNA221, miRNA22, miRNA181a, miRNA181c, miRNA103, miRNA191, let7d and small RNAs (5SrRNA and U6snRNA) that are usually expressed in exosomes.
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Q: Why is the exosomal miRNA level in plasma higher than that in serum?
A: This may be related to the preparation of serum samples. Compared with plasma, clots need to be removed from the whole blood in serum samples. This step may lead to the loss of exosomes, thus reducing the production of miRNA in exosomes. However, there is less chance of losing vesicles in the plasma, so the possibility of losing exosomal miRNA is less.
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Q: How to get pure exosomal miRNA population free of contaminants like miRNA bound to lipoprotein?
A: A large part of miRNA is related to proteins. There are already some efficient isolation kits on the market that can rescue exosomes from other contaminants such as lipoproteins. In addition, it has been proved that pure exosomes can be obtained by a two-step separation (gradient density centrifugation and immunoaffinity capture) method.
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Q: Is circulating miRNA stable in the blood? Why?
A: Although RNA molecules are known to be highly unstable, previous studies have shown that miRNAs are very stable in plasma and serum and resistant to RNase activity, extreme pH, and multiple freeze-thaw cycles. This is because circulating miRNAs are contained in exosomes, which can protect them against RNase activity and make them stable in plasma / serum.
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Q: In general, what is the RNA content of exosomes from different sources?
A: Most exosomes carry very small amounts of nucleic acid. The amount of RNA in exosomes from different sources is also different, which should be determined according to the actual detection. The content of RNA in exosomes from different sources is different, depending on the sample type, sample volume, isolation method and exosome content / concentration. Creative Biolabs has equipped with several powerful techniques including quantitative PCR (qRT-PCR), next generation sequencing (NGS), and RNA microarray for the qualitative and quantitative analyses of exosomal RNA.
In general, compared to samples from other sources (cell culture supernatant, urine, milk, etc.), the level of RNA content in serum / plasma samples is even higher. Even so, the concentration of these RNAs is too low to be seen in agarose gel or even in nano drops. We have seen exosomes derived from 1 ml human plasma typically yielded approximately 1-8 ng of RNA.
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Q: What is the initial volume of plasma/serum required for qPCR or simultaneous exosomes RNA sequencing and protein profiling?
A: 1-2 ml of serum/plasma is sufficient for subsequent qPCR detection; 4ml of serum/plasma can meet the needs of RNA sequencing and protein profiling.
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Q: How do you prepare exosome standards?
A: Creative Biolabs prepares exosome standards using differential ultracentrifugation and microfiltration. Exosomes are then stabilized and lyophilized for long term storage at 4°C. These standards are suitable for positive control in multiple applications (ELISA, WB, FACS, etc.).
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Q: Does the lyophilization affect the shape or the integrity of the vesicles?
A: The lyophilization does not affect the shape or the integrity of the vesicles. Exosomes in lyophilized form can be kept at 4°C up to 3 years.
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Q: How can I store the lyophilized exosome standards after reconstitution?
A: After reconstitution, exosome standards can be stored at -20°C for 1 month and at -80°C for 1 year. Strictly avoid freeze-thaw cycles.
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Q: Why can't I resuspend exosome standards in PBS?
A: Exosome standards are lyophilized in an appropriated buffer which guarantees the appropriate salt concentration, and thus they must be reconstituted in deionized water. Further dilutions must be done by adding 1XPBS, which is compatible with most of the downstream assays, such as protein/RNA extraction, RT-PCR, WB, TEM assay, surface labeling, etc.
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Q: How to prepare samples for WB analysis from lyophilized standards?
A: Reconstitute the lyophilized exosome standards by adding ddH2O to get the final concentration of 1 μg/μL. Add the appropriated quantity of Beta-MercaptoEthanol or DTT if necessary. Do not add Beta-MercaptoEthanol or DTT when analyzing tetraspanins, such as CD63 or CD9. We recommend using 15 μg of exosome standards per lane.
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Q: How can I detect DNA from the Exosome Standards?
A: Exosome contains a very small amount of DNA, and most of DNA is fragmented and single stranded. Therefore, it is suitable for an agarose gel analysis. DNA can be quantified by nanodrop analysis or by qPCR.
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Q: In addition to the particle number and the protein content, do you also characterize their DNA/RNA contents of lyophilized exosome standards?
A: For a new exosome standard lot, we usually do not perform the extraction of DNA or RNA. Lyophilized standards are usually used as a positive control in DNA or RNA kit, so it is possible to use them for isolation of nucleic acid.
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Q: Do you offer products for Extracellular Vesicle (EV) tracking Studies?
A: Creative Biolabs offers highly-quality fluorescent exosome standards for EV tracking studies in fluorescence microscopy experiments. Fluorescent exosomes are labeled with green dye, which provides a stable fluorescent labeling. The excitation maximum of fluorescent exosome standards is 500 nm-650 nm and the emission maximum is 510 nm-665 nm.
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Q: What should I do when WB does not seem to work?
A: There are several typical reasons for this:
1) Insufficient sample volume added. Exosomes contain relatively low protein cargo. For initial experiments, we recommend adding as many samples as possible.
2) Antibodies are not the best. We recommend testing antibodies from 2-3 manufacturers (such as anti-CD63 or other exosome markers) and carefully checking the recommended concentrations. In addition, they should ideally be used fresh, and need to be properly stored.
3) Some gel conditions may be more suitable for target antibodies (such as reduction / non-reduction and denaturation / non-denaturation) according to the exosome surface markers. We recommend that you check with the manufacturer and exosome community what conditions are recommended for the specific marker you are targeting and the specific antibody you are using.
Westerns can be tricky, and we recommend using positive controls for initial testing to verify the entire workflow. Also, try not to use proteins with very high or low concentrations in your sample as markers to prevent the blot from being overloaded or without signal.
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Q: Why can't I see a pellet after centrifuging exosomes?
A: This phenomenon is quite normal. Unless your starting material is a large sample or contains particularly rich exosomes, you will not expect to see precipitation after centrifugation. Exosomal particles are very small and can hardly be seen in an ultracentrifuge tube. Be assured that if you follow the isolation protocol, there will be exosome in the tube.
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Q: If the exosome precipitate is not visible, do I need to leave some supernatant in the test tube to ensure that no exosome precipitate is lost?
A: It is important to completely remove the supernatant. Since the exosome pellet adheres very tightly to the tube, supernatant can be completely removed and resuspended in PBS. If this is not done, there will be a large amount of reagent remaining, and some of them may still be present as aggregates at the bottom of the tube when the exosomes are resuspended in PBS.
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Q: Does the exosomes under electron microscopy have to have a membrane structure? If we see a sphere without membrane structure, can we judge whether it is an exosome?
A: Results from negative staining electron microscopy showed that the morphology of exosomes is saucer-like or pancake-like, with a slightly bright circle on the edge. If the structure is a sphere without a membrane structure, it’s likely to be a lipoprotein particle or protein clusters.
