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View additional product information for Total Exosome Isolation Reagent (from other body fluids) - FAQs (4484453)
76 product FAQs found
For the short-term, exosomes can be stored at 4 degrees C for up to 1 week. For the long-term, exosomes can be stored at -20 degrees C or -80 degrees C. When storing exosomes for the long term, it is important to consider whether they will need to be thawed more than once for the target application. If multiple applications (and thus multiple thaws) will be used for analysis, then we recommend aliquoting the exosome resuspensions into multiple tubes so that each tube will only undergo one freeze/thaw cycle. We have found that multiple freeze thaw cycles can cause damage to the exosomes and reduce their numbers.
Unlike serum, plasma contains numerous clotting factors and some additional proteins that can make it difficult to work with. We‘ve provided two protocol options, one with proteinase K (PK) and one without, in order to ease this difficulty. The protocol using PK is most useful when the end goal is analysis of the RNA or protein cargo contained inside the exosomes. It can also be used to isolate exosomes for use in other downstream applications, but it is most useful for RNA and protein analysis. The protocol without PK also isolates good quality exosomes, just not quite as pure as the PK protocol. The “no PK” protocol is more useful for isolating exosomes that will be used for surface protein analysis or electron microscopy identification.
There are several possible reasons why Western blotting analysis is challenging:
1. Not enough sample volume added. Exosomes can contain a fairly low amount of protein cargo, so for an initial experiment we recommend adding as much of the sample as possible.
2. Antibody concentration should be titrated. Also, they should ideally be used fresh and need to be stored properly.
3. Depending on the exosomal surface marker, certain gel conditions might be more optimal for the target antibody (e.g., reducing/nonreducing and denaturing/nondenaturing). We suggest checking with the manufacturer and exosome community about which Western blotting conditions are recommended for the specific marker you are targeting and the specific antibody you are using.
4. General Western techniques. Westerns can be tricky so we recommend the use of a positive control for initial testing to make sure the entire workflow is functioning as it should. Any protein or antibody can be used as long as they meet the conditions you need (e.g., denaturing vs. non-denaturing). In addition, when picking the protein, try to steer clear of those that are present at very high or very low concentrations in your sample to prevent overloading the blot or total absence of signal.
This can vary depending on the sample type, volume of sample, isolation method, and exosome content/concentration. Listed below are some examples:
1) When exosomes are isolated from 30 mL of HeLa cell culture medium using the Total Exosome Isolation Reagent, it is possible to recover approximately 8 ng exosomal RNA.
2) For exosomes recovered from 4 mL serum, approximately 2 ng exosomal RNA can be obtained.
In both cases, these amounts of RNA are sufficient for RNA library prep for Ion PGM or Ion Proton sequencing. For real-time PCR analysis, substantially smaller amounts of RNA are needed and much lower sample volumes can be used. For example, RNA recovered from 3 µL serum or 30 µL medium is enough for one qRT-PCR reaction.
No, the described effect does not have a negative impact on the RNA recovery.
The Total Exosome RNA & Protein Isolation Kit (Cat. No. 4478545), developed specifically for exosome samples, uses an initial acid-phenol:chloroform extraction followed by a final purification over a glass-fiber filter column to provide a robust initial RNA purification. After the acid-phenol extraction, ethanol is added to the aqueous phase and then passed through a filter cartridge containing the glass-fiber filter, which immobilizes the RNA. The filter is washed, and the RNA is eluted with a low ionic-strength solution.
The kit recovers all RNA longer than approximately 10 nt through several kb, although the majority of RNA contained in purified exosomes ranges in size from 20-300 nt. The kit also contains an additional protocol for enrichment of short RNA (less than 200 nt), but we recommend the total RNA isolation protocol in order to maximize recovery of all RNA, including vario+us mRNA, rRNA, and ncRNA fragments.
This kit also provides an option to recover protein from the same sample through the use of the Exosome Resuspension Solution.
The kit can be used to isolate RNA and protein from exosomes purified from any sample type, using either the Total Exosome Isolation reagents or any other protocol such as ultracentrifugation.
Yes. We have successfully recovered exosomes from up to 5 mL of CSF using the procedure listed in the Total Exosome Isolation (from other body fluids) manual (Cat. No. 4484453).
Yes, overnight precipitation at 4 degrees C is acceptable for urine, CSF, and amniotic fluid without sacrificing quality or yield.
For larger sample volumes than those recommended in the manuals, a longer centrifugation is recommended to ensure maximum recovery of the exosomes. The exact time will depend on several factors including the rotor, the sedimentation coefficient of the exosomes, size of the tube, sample volume and type, and centrifugation speed, and should be determined empirically. For example, for 5-10 mL sample volumes, 1 hr of centrifugation at 10,000 x g is sufficient to pellet the exosomes.
Here are some examples:
HeLa cells grown to approximately 2 x 10e7 per T175 flask in 30 mL medium with exosome-depleted FBS. From 1 mL of this medium, one can recover approximately 4-8 x 10e9 exosomes using the Total Exosome Isolation Reagent (from cell culture media), as measured with Nanosight LM10 instrument. These numbers will be somewhat different for different cell lines and whether exosome-depleted FBS or no FBS or synthetic medium is used.
From 100 µL serum one can recover approximately 1.5-3 x 10e11 exosomes using Total Exosome Isolation Reagent, as measured with Nanosight LM10 instrument.
Here is a paper put together by our scientists that compares our exosome isolation protocol with standard ultracentrifugation protocols, with respect to RNA profiling: https://www.wjgnet.com/2222-0682/full/v3/i1/11.htm
For each reagent, the minimal volume tested can be found listed in the reagent manual. For most body fluids the minimum volume tested is 100-200 µL, but it is slightly larger for urine (800 µL) and cell culture media (1 mL). Smaller volumes can be used, especially for serum and plasma, but we‘ve found that the minimums listed in the manuals and above provide a usable amount of exosomes for multiple downstream applications.
Serum and plasma contain a very high number of exosomes, thus the pellet is visible even if you isolate exosomes from as little as 100 µL. Other body fluids, such as urine or cell culture media, have significantly lower concentrations of exosomes, and the pellet is often not visible after centrifugation. Since the pellet sticks very tightly to the tube, it is okay to remove the supernatant completely prior to resuspending in PBS. If needed, marking the tube so that you know where the pellet will adhere upon centrifugation. It is absolutely crucial to remove the supernatant completely. If you don‘t, there will be a significant amount of the reagent left, and when you resuspend the exosomes, some of them might still be in the form of aggregates at the bottom of the tube.
The reagent does not bind the exosome surface, and only trace amounts remain in the exosome pellet after isolation, so it should not interfere with downstream biological studies. However, it is important to remove the supernatant completely, prior to resuspending the exosome pellet in PBS or other buffer of choice. In case there are still concerns regarding trace amounts of the reagent being present, they can be removed by dialysis or using Exosome Spin Columns (MW 3000) (Cat. No. 4484449).
We currently have a total of five reagents that allow isolation of exosomes from major body fluids: serum, plasma, urine, “other body fluids” (CSF, saliva, milk, ascitic fluid, amniotic fluid), and cell culture media. All of these reagents share the same core compound, but they and their protocols have been carefully optimized to enable efficient isolation of exosomes from the specific sample type.
Plasma is a more challenging type of sample compared to serum, as it has high levels of clotting factors, which can be more problematic. The current serum reagent will work on plasma, but the preparation will likely contain more contaminating proteins and microvesicles. For plasma, we recommend using the Total Exosome Isolation reagent (from plasma) (Cat. No. 4484450), as the reagents and protocol have been specifically optimized to handle plasma and its different composition.
Yes, the reagents can also be used with mouse samples. Presumably, the reagents can also isolate exosomes from samples of any species, but they have only been tested with human and mouse.
The obtained sample contains all exosomes, with insignificant amounts of some other microvesicles, and large protein molecules/complexes that have been co-precipitated (in the case of serum and some of the other body fluids). This purity level works fine for most applications and is balanced by the method benefits which include a fast and simple workflow, no need for special equipment (such as an ultracentrifuge), complete recovery of exosomes, flexibility to work with small sample volumes (e.g., 100 µL), and the capability to process multiple samples in one experiment.
For ultra-pure exosomes from cell culture media, we have available Dynabeads magentic beads decorated with anti-CD63 antibodies, which allow recovery of a very clean population of exosomes, following initial purification with the Total Exosome Isolation Reagent (from cell media). Workflow time will increase and the final yield of the exosomes will be lower, but for projects that require an ultra-clean population of CD63-positive exosomes from cell culture media this is the best option.
Dynabeads magnetic beads complexed with streptavidin (Cat. No. 10608D) are also available for use with a customer‘s own biotinylated antibodies specific for their exosome sub-population of choice. These products can be used not only for isolation of highly pure exosome sub-populations, but also allow detection of exosomes with flow cytometry, something that has been extremely difficult to achieve due to their small size.
We believe that these products are currently the best options for exosome isolation, but as the definition of exosomes solidifies and the demands of the field of microvesicle research become clearer, we can focus on additional products
Isolation of exosomes is presently a tedious, non-specific, and difficult process. In the course of development of reagents for isolation of exosomes, we evaluated many different technologies, including “gold standard” ultracentrifugation, ultrafiltration; gel-filtration columns, HPLC, and filters. In addition to these simpler methods, we evaluated more advanced approaches including precipitation using various polymers, and bead and column binding using antibodies and various lectins. We also evaluated commercially available products from System Biosciences and other companies. After evaluation, we selected one of the polymers, based on its superior performance, which became the key component of the Total Exosome Isolation reagents (patent application filed). By tying up water molecules, the reagent forces less-soluble components, such as vesicles, out of solution, which allows them to be collected by a short, low-speed centrifugation. The recovered exosomes are then ready for either biological studies or end-point analysis.
Traditional isolation of exosomes from cell culture media and body fluids is a tedious and difficult process with the most widely used approach based on ultracentrifugation in combination with sucrose density gradients or sucrose cushions to float the relatively low-density exosomes away from other vesicles and particles. These protocols can range in time from 8 to 30 hrs and require an ultracentrifuge and extensive training to ensure successful isolation of exosomes. Despite these drawbacks, ultracentrifugation is still the most popular approach for exosome isolation. However, within the last 2 years, several exosome isolation reagents for cell culture media and various body fluids have become commercially available. Thermo Fisher Scientific Total Exosome Isolation reagents allow recovery of exosomes using a very short and reliable protocol which is becoming more and more popular.
In addition to their 30-150 nm size, to be categorized as exosomes, currently, the vesicles should be positive for certain surface protein markers, such as tetraspanins. The most widely accepted marker is CD63, but CD81, CD9 are utilized as well. Western blotting for these targets on the sample of interest is a relatively simple way to confirm the vesicles are exosomes. However, the current definition of exosomes is not set in stone as there is no absolute consensus in the field. It will probably take another several years for the field to agree on the exact specification and nomenclature for all nano- and micro- vesicles including exosomes.
At the moment there are only two straightforward options to determine the concentration of exosomes in a sample: the NanoSight instrument and the Izon instrument. The Nanosight instrument enables counting and sizing of nanoparticles (10-1000 nm size) using light scattering and brownian motion, while the Izon instrument accomplishes the same thing using nanopore analysis.
Exosomes, at 30-150 nm, are too small to be seen using a regular microscope, which is limited to objects that are at least several micrometers in size. The typical methods of analysis for size distribution include the Nanosight instrument and electron microscopy. Although very different in methodology, both technologies allow one to study nanoparticles as small as 10 nanometers in size.
Exosomes are classically described as vesicles originating from the endosomal compartment through fusion of multivesicular bodies with the plasma membrane. They are a part of a larger family of vesicles secreted by cells, including microvesicles, ectosomes, and shed particles, which originate by direct budding from the plasma membrane. It is extremely challenging to separate these entities using currently available techniques and instruments due to overlap in their size, density, and overall similar composition.
Exosomes are tiny vesicles (30-150 nm) containing protein and/or RNA cargo within a lipid bi-layer membrane. Exosomes can differ extensively in both their cargo and surface proteins, and different cell types can secrete different, sometimes multiple, types of exosomes.
The current definition of exosomes is complex as no absolute consensus has been made in the research field. Typically, exosomes are defined as vesicles floating in sucrose solution at a density of approximately 1.13 to 1.19 g/mL during ultracentrifugation-based isolation with an expected size of 30-150 nm based on electron microscopy analysis. Exosomes can also be defined and identified by their surface protein markers, which include: tetraspanins (CD63, CD81, CD9) and others like ALIX. Currently, we don‘t have the appropriate tools or enough knowledge to create a clear and simple definition of exosomes that would differentiate them from other micro/nanovesicles.
Ultracentrifugation typically recovers fewer exosomes compared to the Total Exosome Isolation reagent. Since cell culture medium has very low exosome content, the exosome pellet is invisible in many cases. Assuming that you have not lost the pellet, you can do Nanosight analysis or use some other easy readout to confirm how much you have isolated.
Sometimes exosomes stick together upon isolation with the reagent or by ultracentrifugation. They don‘t fuse, but are just clumped together. You can (1) add buffer such as PBS to the exosome pellet, let the sample sit at RT for several hours, then pipet the sample extensively up and down, or (2) gently vortex the pellet.
We offer the Total Exosome RNA and Protein Purification Kit (Cat. No. 4478545) for isolation of total RNA and protein from exosomes.
We typically count exosomes with the Nanosight LM 10 instrument, rather than measure their weight. To provide an idea about numbers, when HeLa cells were grown to approximately 2 x 10e7 cells per T175 flask in 30 mL cell culture medium in the presence of exosome-depleted FBS, 1 mL of this cell medium yielded approximately 4-8 x 10e9 exosomes isolated with the reagent. The number will be somewhat different depending on the cell line or medium used.
We recommend using the Qubit 2.0 Fluorometer to easily measure the signal associated with exosomes post-labeling.
Please review the following options:
- You can normalize samples using miR-16, miR-24, 18S rRNA, or GAPDH, which are all highly abundant in serum-derived exosomes.
- If you have enough biological replicates and a reliable workflow (sample prep to RT to qPCR) and minimal error bars, you can skip normalization and instead, use the same sample volume input.
We recommend resuspending the exosomes in PBS. Ideally, the isolations should be performed with a lower volume of serum, e.g., 100 µL, so that the exosome pellet is small and easier to resuspend. This sample should contain a substantial amount of RNA and protein for typicaly downstream assays, including Western blotting and Real-Time PCR. If the pellet is large and difficult to resuspend, (e.g., if the isolation was done from greater than 1 mL of serum), let it sit in PBS for 1 hour at room temperature followed by pipetting or gently vortexing.
In our experience, there is no difference between exosome isolation from fresh serum and frozen serum, stored at -20 degrees C or -80 degrees C. However, multiple (greater than 5) freeze/thaw cycles can damage the exosomes.
The Total Exosome Isolation reagent precipitates exosomes by tying up water molecules and forcing them out of solution. Once the reagent is completely removed and PBS is added, exosomes will start going back into solution. Typically, no washing is needed. You can collect the remaining droplets of the reagent by addtiional quick centrifugation, and discard them. To do a wash, we would recommend performing a quick rinse in PBS by spinning for 5 minutes at 14,000 g at 4 degrees C. We do not recommend vortexing of the pellet.
The easiest way to count exosomes is by Nanosight analysis (e.g., Nanosight LM10). With most of these instruments, you can obtain valuable information on their size distribution and count. With electron microscopy, you will be able to see just a few “zoomed in” vesicles and see their size and shape, but you will not be able to quantify exosomes.
There are typically no problems with contamination when using exosomes (recovered with the Total Exosome Isolation reagent) in downstream biological assays.
We would recommend using 20-50 mL of cell culture medium and 4-8 mL of serum or plasma. You can use the Total Exosome Isolation reagent to recover the exosomes. Ultracentrifugation is also an option, but yields will be lower.
Yes, exosomes have a very diverse RNA and protein content. RNA species, in addition to mRNA, include miRNA, rRNA, tRNA, and many non-coding RNAs, some of which do not map to the databases. Most of the exosomal RNA “cargo” is short, 20-200 nt (including mRNA fragments), but some mRNA molecules are full-length, up to several kb long.
No, exosomal RNA has about the same stability as RNA recovered from other sources. One important thing to note: since exosomes contain very low amounts of RNA, the concentration of isolated exosomal RNA in the tube is often is very low, resulting in very poor stability. We recommend taking extra precaution to avoid RNase contamination by using RNase-free water, buffers, tips, tubes, etc., and storing the RNA stock at -20 degrees C or below, in aliquots, at the highest possible concentration.
When you harvest the cell culture medium, it should be spun down to remove cells and debris. You can then keep this “clarified” medium at 4 degrees C for up to one week, or isolate exosomes by precipitation with the Total Exosome Isolation reagent or other techniques.
We recommend using our Total Exosome RNA and Protein Isolation Kit (Cat. No. 4478545), which has reagents enabling splitting of the exosomes and recovery of either their protein cargo or RNA cargo (via column based purification). Overall, exosomes are similar to cells in terms of membrane composition, and a number of commonly used sample prep kits will work fine on exosomes as well.
We recommend using anti-mouse IgG HRP, since it will not recognize the heavy or light chains of the antibodies eluted off the beads.
Exosomes can be vortexed briefly without damaging their membrane, but there are no detailed studies on the effects of prolonged vortexing or sonication on exosome integrity.
We would not recommend multiple (greater than 5) freeze/thaw cycles, as that would damage the exosomes. Ideally, we would recommend freezing the exosomes in single-use aliquots.
Prior to Western blotting, exosomes can be pre-enriched followed by lysis using lysis buffers such as RIPA, NP40, or Triton, together with protease inhibitors. This is followed by a short vortex or sonication in order to have complete lysis. For detection by Western blotting, the pre-enriched exosomes should be lysed in 5X RIPA, for example. We have tried the following: Mix 7.5 µL pre-enriched exosomes + 1.9 µL 5X RIPA w/ protein inhibitors, followed by a short sonication and incubation on ice for 15 mins. Take 9.4 µL lysed exosomes + 9.4 µL 2X SDS (Laemmli) + 1 µL loading buffer and incubate 5 min at 95 degrees C and add all to one well. Given that the pre-enrichement step was successful, this should ensure maximum loading of exosomes on the gel and detection by Western blotting.
If the pre-enriched exosomes have been further isolated using magnetic beads to pull out subpopulations of exosomes, these can also be lysed using 1X RIPA on ice followed by Laemmli and loading buffer in the same way. The beads can either be removed prior to gel loading using a magnet or apply the lysed exosomes and beads on the gel. The beads will be left in the well while the target moves in the gel.
Please see our protocols, including Western blotting:
- Schageman J., Zeringer E., Li M., Barta T., Lea K., Jian Gu, Magdaleno S., Setterquist R., and Vlassov A.V. The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. BioMed Research International. 2013, 2013:253957. doi: 10.1155/2013/253957.
- Zeringer E., Li M., Barta T., Schageman J., Pedersen K.W., Neurauter A., Magdaleno S., Setterquist R., Vlassov A.V. (2013) Methods for the extraction and RNA profiling of exosomes. World J Methodology. 3, 11-18.
While different cells react differenty under different conditions, in general, if you grow cells in FBS-free media, fewer exosomes will be secreted as cells are starved. Some cells, such as HeLa cells, are more durable under exosome-depleted FBS conditions. We have been able to isolate 4-8 x 10E9 exosomes from 1 mL of this cell culture medium. For more information, please refer to the following reference: Schageman J. et al., BioMed Research International, Volume 2013 (2013), Article ID 253957.
A pure sample of exosomes contains very low amounts of RNA. Therefore, the best way to increase yeild is to scale up the isolation. For cell culture media, you can try starting with 100 mL of sample.
Yes, please check our application note at https://tools.thermofisher.com/content/sfs/brochures/Exosome%20Tracing_App%20Note.pdf that lists protocols employing SYTO RNASelect stain (Cat. No. S32703).
There are several typical reasons why Western blot analysis does not work:
1. Not enough sample volume added. Exosomes can contain a fairly low amount of protein cargo, so for an initial experiment we recommend adding as much of the sample as possible.
2. Antibodies are not optimal. We suggest testing antibodies (e.g., anti-CD63 or other exosomal marker) from 2-3 manufacturers, carefully checking what concentration is recommended. Also, they should ideally be used fresh, and need to be stored properly.
3. Depending on the exosomal surface marker, certain gel conditions might be more optimal for the target antibody (e.g., reducing/nonreducing and denaturing/nondenaturing). We suggest checking with the manufacturer and exosome community about which Western blot conditions are recommended for the specific marker you are targeting and the specific antibody you are using.
4. General western techniques. Westerns can be tricky, so we recommend the use of a positive control for initial testing to make sure the entire workflow is functioning as it should. Any protein or antibody can be used as long as they meet the conditions you need (e.g., denaturing vs. non-denaturing). In addition, when picking the protein, try to steer clear of those that are present at very high or very low concentrations in your sample to prevent overloading the blot or total absence of signal.
You can add the loading buffer directly to the exosome pellet, heat, and load onto your gel for downstream western analysis. There is no need to air dry the pellet. 1 mL cell culture medium per lane is a good starting point, but may need to be optimized based on the protein of interest, antibody used, etc.
When you isolate exosomes from cell culture medium, the pellet is invisible in most cases, unless you are using a large volume (e.g., 30 mL). With serum, plasma, and other body fluids, the pellet is typically visible if you process over 100 µL of sample.
While we have not tested ESC, for those media that we have tested, the number of secreted exosomes and isolation protocols are very similar. When growing cells, use chemically defined media or exosome-depleted FBS. Exosomes can be collected at 24 hours. 1-5 mL of media is a good starting point to isolate exosomes. This should be enough for several real-time PCR or Western blotting experiments. Sequencing and some other downstream analyses may require more starting material, and for this, we would recommend 30 mL cell culture medium to start.
Please refere to the following reference: The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. Jeoffrey Schageman, Emily Zeringer, Mu Li, Tim Barta, Kristi Lea, Jian Gu, Susan Magdaleno, Robert Setterquist, and Alexander V. Vlassov. Hindawi Publishing Corporation, BioMed Research International. Volume 2013, Article ID 253957, 15.
Overall, we have not encountered issues related to hemolysis. We would recommend using care with body fluid collection and preservation. For recommendations on standardization of sample collection, isolation, and analysis, please refer to the following reference: Witwer et al., Journal of Extracellular Vesicles (2013) 2:20360.
Overall, the exosomes recovered from cell culture media are very clean. Exosomes derived from body fluids sometimes have a few contaminating microvesicles and large protein aggregates. The recovered exosomes can be analyzed by Nanosight (size distribution and count) or electron microscopy. Western analysis could be performed using protein markers, including CD63, CD81, CD9, Alix, or Annexin. RNA cargo can be analyzed by real-time PCR (e.g., of let7).
We routinely use the Total Exosome RNA and Protein Isolation kit (Cat. No. 4478545), but there are a number of other kits that are popular among researchers, including mirVana isolation kits and TRIzol reagent. For analysis, Real-Time PCR with TaqMan miRNA assays would be our recommendation.
Apoptotic bodies are large (greater than 800 nm), so they will mostly be removed from the sample during pre-spin (along with cells and debris). The Total Exosome Isolation reagent is added at the next step, and it precipitates primarily exosomes (30-150 nm).
We have not optimized and validated a protocol to use the CD63 isolation/detection reagent with plasma samples. However, there are references in the literature that use this product in plasma samples:
Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study | PLOS ONE
Yes, our Total Exosome Isolation reagents enable precipitation of the entire exosome population from cell media and all body fluids. They should also work on any kind of supernatant that contains exosomes.
We would suggest looking at the exosomes at the ultrastructural level using epon embedding, sectioning, and electron microscopy.
Yes, the exosomes recovered with the Total Exosome Isolation reagent (Cat. No. 4478359) are fully intact, free, not aggregated, and functional. They can be used for any downstream biological experiments, such as tracing them in cells or any functional studies.
The pellet is soluble in PBS or any other buffer. It is very easy to resuspend exosomes recovered from a small sample of cell culture medium (e.g., 1 mL), but if you are processing a large sample of plasma or serum, the pellet will be pretty large. In this case, you can add buffer, let it sit for 30 min at 37 degrees C, then vortex gently or pipet up and down to resuspend the exosomes.
By Nanosight analysis, most of the recovered exosomes are in the expected size range, 30-150 nm.
You can try to separate them by size, since apoptotic bodies are approximately 800-5000 nm, while exosomes are approximately 30-150 nm.
We have standardized dissolving the pellet of cell culture exosomes after isolation with Total Exosome Isolation reagent as follows:
Beckman J2-21M/E centrifuge with JA20 rotor
Nalgene centrifuge tubes for Beckman centrifuge
PBS, 0.22 µm filtered
- Start with 20-25 mL of cell culture supernatant from overnight isolation (using Total Exosome Isolation reagent).
- Centrifuge at 10,000 x g = 9200 rpm for 1 hour (set temperature at 10 degrees C, as lower setting may result in temperature as low as 0-2 degrees C during centrifugation).
- Remove supernatant by suction.
- Leave tubes upside-down on absorbant paper for 10 min at RT to remove residual buffer.
- Add buffer to cover the pellet when the tubes are placed at an angle to the bench (500 µl PBS/sample tube), leave for 30 min at RT.
- Carefully resuspend by pipetting and pool sample from each centrifuge tube.
- Collect residual volumes from each sample tube by centrifugation for 8 min at 350 x g.
- Aliquot and freeze at -80 degrees C.
Exosomes can be processed for electron microscopy for ultrastructural analysis both prior to Dynabeads magentic beads isolation and after isolation.
Prior to isolation the exosomes pool can be immunolabeled and processed for negative stain prior to ultrastructural analysis. Such a protocol could look like this:
- Load exosomes undiluted at RT for 15 min.
- Block with 0.5% BSA for 10 min.
- Label with primary antibody for 30 min.
- Wash 5X with PBS for 10 min total.
- R&M (1:100) for 30 min.
- Wash 5X with PBS for 10 min total.
- Prot A Au 10nm for 15 min.
- Wash 5X with PBS for 10 min total.
- Wash 5X with water for 10 min total.
- Embed in 0.3% uranyl acetate in methyl cellulose.
- Conduct TEM analysis.
Subpopulations of exosomes prepared using the Total Exosome Isolation kit or ultracentrifugation can also be processed for ultrathin sectioning and electron microscopy. After isolation and washing of the exosomes on the surface of the Dynabeads magnetic beads, they can be processed using the traditional TEM protocol described by Pedersen et. al. in J Virol (1999) 73:2016–2026. In brief, for conventional Epon embedding and sectioning, Dynabeads magnetic beads with exosomes can be fixed for 1 hr in 1% glutaraldehyde in 200 mM cacodylate buffer (pH 7.4), washed repeatedly in aqua destillata, and incubated for 1 hr in cacodylate buffer containing 1% OsO4 and 1.5% K3Fe(CN)6. Following two subsequent 30-min incubations in 1% tannic acid and 1.5% magnesium uranyl acetate, the samples are dehydrated by using ethanol and embedded in Epon. Ultrathin sections can be stained with lead citrate.
While we have not tested this, the Total Exosome Isolation reagent for use with cell culture medium should work, since tears are similar to cell culture medium in many ways (e.g., non viscous, with low exosome content).
Since exosomes are in the same size range as many viruses, visualization of exosomes by TIRF microscopy should work. For ultrastructural analysis, one would need to increase the resolution and use electron microscopy.
The current definition of exosomes is sophisticated and there is no consensus in the field. Exosomes are typically defined as vesicles floating in sucrose solutions at a density of approximately 1.13 to 1.19 g/ml during ultracentrifugation-based isolation. Expected size is 30-120 nm, based on the electron microscopy analysis. Protein markers include: tetraspanins (CD63, CD81, CD9), and some others. But everyone agrees that at the moment we don‘t have enough knowledge and appropriate tools to set a clear and simple definition of exosomes and other micro/nanovesicles.
There are some variations to the ultracentrifugation protocols, not based on the cell lines used so much as the experience in that particular lab, including what G-force they recommend, duration of ultra-centrifugation, straight sedimentation vs cushion vs sucrose gradients to obtain the top quality exosomes at reasonable yields. We often recommend protocols developed by Clothilde: C. Thery, S. Amigorena, G. Raposo and A. Clayton “Isolation and characterization of exosomes from cell culture supernatants and biological fluids.” Curr. Protoc. Cell. Biol., Chapter 3, Unit 3: 22, 2006.
The Total Exosome Isolation reagent allows recovery of the entire exosome population from the sample, in contrast to ultracentrifugation protocols, which recover significantly less material. Thus, the pellet is larger. The purity of exosomes is very similar for cell media samples (reagent vs ultra); for more challenging samples such as serum (which are more viscous and have much higher protein content, etc.) the reagent recovers exosomes at a lower purity (few microvesicles and large protein complexes co-precipitate) compared to ultracentrifugation. Having said this, you can obtain enough material for all standard types of downstream analysis, such as qRT-PCR or Western blot with either method.
Please review the references below for examples of exosomal markers used for this purpose:
- Journal of Extracellular Vesicles (2013) 2:20360.
- EMBO J (2007) 26(5):1221–1233.
- BMC Cancer (2010) 10(1):294.
- Cell (2012) 151:1542-1556.
When you are isolating exosomes/extracellular vesicles from cell media, for example, or body fluid such as blood, the first step is gentle centrifugation, in order to get rid of cells and cell debris. Whatever remains in the supernatant is exosomes, microvesicles, and proteins. Then you can recover exosomes using the Total Exosome Isolation reagents, and, if required, obtain an ultra-pure population of CD63 positive exosomes with CD63-coupled magnetic beads (Cat. No. 10606D). You can also isolate CD9 positive exosomes using CD9-coupled magnetic beads (Cat. No. 10614D), CD81 positive exosomes using CD81-coupled magnetic beads (Cat. No. 10616D), or use magnetic beads with EpCam (Cat. No. 10618D) to target EpCam-positive exosomes. You are right that it's not trivial (at least at the moment) to capture the specific populations using some antibodies (or other affinity reagents) since there are no absolutely specific markers for exosomes or other vesicles. It's not a problem for many projects, unless you suspect your cells are “leaky” and release some components into the media. CD63, CD81, CD9, Alix, Annexin, and TSG are not strictly specific exosome markers.
Plasma is a more challenging type of sample compared to serum. It has rather high levels of clotting factors. The current serum reagent will work on plasma, and it will precipitate all exosomes. However, the preparation will have some contaminating proteins and microvesicles, which will work for some projects, but not be acceptable for others. We recommend using our specifically optimized kits for recovery of exosomes from blood plasma (Cat. No. 4484450), as well as urine (Cat. No. 4484452), or other body fluids (Cat. No. 4484453).
The reagent for exosome isolation from serum can be used with serum from any species in addition to human. When working with small volumes of non-human samples (e.g., mouse) or when it is desired to maximize the recovery of exosomes, we recommend following the standard protocol except for spinning the samples (after incubation with the reagent) to precipitate the exosomes at 4 degrees C.