Q: What is the current method for exosome isolation?

A: Traditional isolation of exosomes from cell culture media and body fluids is a tedious and difficult process, typically 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 take up to 30 hours, require an ultracentrifuge and extensive training. Despite these drawbacks, this is still the most typical approach for exosome isolation.

However, within the last couple of years, several reagents for isolating exosomes from cell culture media and various body fluids have been made commercially available from Life Technologies and other companies. The Total Exosome Isolation reagents available from Life Technologies allow for the recovery of exosomes, using a very short and reliable protocol. This approach is becoming more and more popular.

 

 

Q: Which products for exosome research are available from Life Technologies?

A: A number of products developed specifically for exosomes research are available, as described here: www.lifetechnologies.com/exosomes. We are continuously expanding this product portfolio.

In addition, we offer many reagents, kits and instruments for analysis of exosomal cargo including: RT-qPCR with TaqMan assays, next generation sequencing on Ion Torrent PGM, Proton and SOLID for RNA and Western blotting instruments and reagents for protein research.

 

 

Q: How do the Total Exosome Isolation reagents work, and what makes them different from the other techniques for exosome isolation?

A: In the course of development of reagents for isolation of exosomes we evaluated many different technologies (e.g. ultracentrifugation, ultrafiltration, gel-filtration columns, HPLC, and filters). We also evaluated more advanced approaches (e.g. precipitation using various polymers, and bead and column binding using antibodies and various lectins). In addition, we evaluated commercially available products from different companies.

We then 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. All Total Exosome Isolation reagents share the same core compound, but have each been carefully optimized (incl. adjustments in protocols), to enable efficient isolation of exosomes from specific sample types.

 

 

Q: Do the Total Exosome Isolation reagents allow isolation of exosomes from other species?

A: Yes, the reagents can also be used with mouse samples.  The reagents can potentially also isolate exosomes from samples of any species, but have so far only been tested with human and mouse.

 

 

Q: Does the Total Exosome Isolation (from serum) reagent work on plasma samples?

A: Plasma is a more challenging type of sample compared to serum, as it has high levels of clotting factors. 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 (from plasma) reagent, with a protocol specifically optimized to handle plasma.

 

 

Q: What is the purity of the exosomes recovered with the Total Exosome Isolation reagent, is it similar to ultracentrifugation?

A: The obtained sample contains all exosomes, with insignificant amounts of some additional microvesicles and large protein molecules/complexes that have been co-precipitated (in 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. The benefits include: 1) A fast and simple workflow, 2) No need for special equipment (such as an ultracentrifuge), 3) Complete recovery of exosomes, 4) The flexibility to work with small sample volumes (e.g. 100μl), 4) The capability to process multiple samples in one experiment.

To allow for the recovery of a very clean population of exosomes following an initial purification with the Total Exosome Isolation (from cell media) reagent, we also offer Dynabeads® coupled with anti-CD63 antibodies. This additional step will increase workflow time, 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. Streptavidin-coupled Dynabeads® are also available for use with your choice of biotinylated antibody - specific for a specific exosome sub-population.

These products can be used not only for isolation of highly pure exosome sub-populations, they also allow detection of exosomes with flow cytometry - something that has otherwise been extremely difficult to achieve due to their small size.  We believe that these products are currently the best options for exosome isolation.

 

 

Q: How do the Total Exosome Isolation reagents compare to the ExoQuick™ reagents from System Biosciences?

A: The Total Exosome Isolation reagents have a unique formulation (patent application filed). Carefully optimized reagents allow recovery of exosomes from major body fluids and cell media. The yield and purity of the exosomes recovered with the products supplied by Life Technologies is equivalent – and sometimes improved - compared to ExoQuick™ reagents. In addition,  the Life Technologies reagents enable isolation of the total exosome population from more sample types, and the products are part of a larger, complete workflow solution from isolation to characterization to in vivo studies.

 

 

Q: What is the minimal volume of starting sample I can use to isolate exosomes?

A: For each reagent, the minimal volume tested can be found listed in the product manual.  For most body fluids the minimum volume tested is 100-200μl, and slightly larger for urine (800μl) and cell culture media (1ml).  Smaller volumes can be used - especially for serum and plasma - but we’ve found that the minimums listed here provide a usable amount of exosomes for multiple downstream applications. 

 

 

Q: Can I isolate exosomes from more than 1 ml CSF?

A: 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.

 

 

Q: If I recover exosomes with the Total Exosome Isolation reagent from a larger sample volume, >5 mL, will I need to spin them for a longer time?

A: 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. For example, for 5-10 mL sample volumes, 1 hour of centrifugation at 10,000 x g is sufficient to pellet the exosomes. But if the sample volume is larger, or centrifuge cannot generate 10,000 x g – longer centrifugation times should be used.

 

 

Q: How many exosomes can be isolated from cell media and serum?

A: The numbers will be somewhat different for different cell lines, and depending on whether you add exosome-depleted FBS or no FBS or synthetic media. Here’s an example: HeLa cells grown to ~ 2 x 107 per T175 flask in 30 ml cell culture media, with exosome-depleted FBS. From 1ml of this cell media, one can recover ~4-8 x 109 exosomes with the Total Exosome Isolation (from cell culture media) reagent, as measured with NanoSight LM10.  From 100 μl serum you can recover ~1.5-3 x 1011 exosomes – with the Total Exosome Isolation (from serum) reagent.

 

 

Q: There are two protocol options for plasma samples, which one should I choose?

A: Unlike serum, plasma contains numerous clotting factors and some additional protein which can make the sample difficult to handle.  To ease this difficulty , we’ve provided two protocol options: One with Proteinase K treatment , and one without.  The protocol including Proteinase K treatment is most useful when the end goal is analysis of exosomal RNA or protein cargo. This protocol can also be used to isolate exosomes for use in other downstream applications, but is most useful for RNA and protein analysis.  The protocol without Proteinase K treatment also isolates good quality exosomes, just not quite as pure as the protocol with Proteinase K treatment.  The protocol without Proteinase K treatment is more useful when isolating exosomes that will be used for surface protein analysis or electron microscopy identification.

 

 

Q: Due to time constraints, I cannot finish the protocol in a single day. Can I precipitate my exosomes overnight?

A: Yes, overnight precipitation at 4° C is acceptable for urine, CSF, and amniotic fluid using the Total Exosome Isolation (from other body fluids) reagent - without sacrificing quality or yield.

 

 

Q: Is there any reagent left in the exosome preparation after isolation? If so, can this interfere with the downstream biological studies?

A: The reagent does not bind to the surface of the exosomes, and only trace amounts remain in the exosome pellet after isolation. This should not interfere with downstream biological studies.  However, it is important to completely remove the supernatant prior to resuspending the exosome pellet in PBS or other buffer of choice. If you still have concerns about trace amounts of the reagent, this can be removed by dialysis or using the Exosome Spin Columns (MW 3000).

 

Q: Is it OK if the exosome pellet is not always visible after isolation? Can we leave some supernatant in the tube, just to be sure not to lose any of the exosome pellet?

A: Serum and plasma contain a very high number of exosomes, thus the pellet is visible even if you isolate exosomes from as little as 100μl. Other body fluids, such as urine, or cell culture media, have significantly lower concentration of exosomes, and the pellet is often not visible after centrifugation. Since the pellet sticks very tightly to the tube, it is OK to remove the supernatant completely prior to resuspending in PBS. If needed, it is easy to label the tube so 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 be still in the form of aggregates, at the bottom of the tube.