Lyophilized Exosome Standards

Lyopihlized Exosome Standards

What are exosomes?

Exosomes are extracellular lipid nanovesicles, in the order of 30-120 nm, secreted by cells and found in all body fluids such as plasma, urine, serum, saliva and cerebrospinal fluid (CSF), etc.

What is the difference between exosomes and microvesicles?

Exosomes are smaller in diameter than microvesicles (30-120 nm vs 100-1000 nm). They have different protein markers and play different roles in cellular communication and genetic exchange.

How Exosome Standards are prepared?

Exosome standards are prepared by differential ultracentrifugation and microfiltration. Exosomes are then stabilized and lyophilized for long term storage at 4°C. Exosomes are suitable for positive control in multiple applications (WB, ELISA, FACS, EM etc.).

Does the lyophilization affect the shape or the yield of the vesicles?

The lyophilization does not affect the shape or the integrity of the vesicles. Exosomes in lyophilized form are stable up to 36 months stored at 4°C.

How can I store the lyophilized Exosome Standards after reconstitution?

Once reconstitute Exosome Standards can be stored at -20° C for up to 1 month and at -80°C for up to one year. Strictly avoid freezing and thawing cycles.

Why I have to resuspend Exosome Standards in deionized water and not in PBS?

When lyophilized, Exosome Standards are in an appropriated buffer which guarantees the appropriate salt concentration and they must be reconstituted in milliQ water. Further dilutions has to be done by adding PBS 1X, which is compatible with most of the downstream assays including RNA/protein extraction, QRT PCR, TEM assay, western blotting, surface labeling, etc.

What are Plasma Exosome Standards?

Plasma exosome standards are purified lyophilized exosomes isolated from a pool of healthy certified donors by differential ultracentrifugation and microfiltration.

How to do you prepare samples for western blot analysis from Lyophilized standards?

We recommend to use 15-20 ug of purified exosomes per lane. Reconstitute the lyophilized exosome standards by adding deionized water (MilliQ) to get the final concentration 1 ug/ul (for 100 ug vials add 100 ul of MilliQ water). Add the appropriated quantity of Beta-MercaptoEthanol or DTT if required.  Not reducing conditions are necessary for the analysis of tetraspanins, such as CD63 or CD9 (don't add Beta-MercaptoEthanol or DTT). Load the samples on the gel for running the SDS-PAGE and WB.

Does cell death affect exosomes?

Yes, cell death definitely affects exosomes secretion. Thus, we always recommend healthy and viable cells for studying exosomes. Additionally, apoptotic cells release a lot of apoptotic vesicles.

What should be the exosome yield per cell?

The exosome yields per cell or the total number may depend on several factors, including the cell type, cell confluence, the culture conditions (growth factors etc), the time of harvest, the uniformity in size of exosomes, and the source type. Thus, one cannot fix a number of exosome yield per cell.

How do I visualize apoptotic bodies, microvesicles and exosome pellet from centrifugation?

To characterize individual extracellular vesicles, you can use western blots (WB), flow cytometry (FACS), global proteomic analysis including mass spectrometry techniques or Electron microscopy and Atomic force microscopy for characterizing individual EVs. For larger vesicles including apoptotic bodies, Cytospins and Immunofluorescent images can be used and for size distribution, Nanosight, Dynamic Light Scattering can be used.

What is the best storage condition of conditioned media (at 4°C/-20°C/-80°C) for subsequent isolation of exosomes?

We recommend processing fresh samples, but cell media can be stored at 4°C for up to one week with no major changes with exosomes/EV being observed. For long term storage -80°C is recommended.

Can I freeze purified exosomes after isolation? Does the freezing affect the exosome structure?

Ideally, exosomes should be processed fresh. Fresh samples are always preferred over frozen samples. However, -80°C C frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple times (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods. The exosomal membrane is different from the cell membrane as it originates from the endosomal membrane and 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 and in size determination studies including dynamic light scattering (DLS), nano tracking analysis (NTA), bioassays and electron microscopy (EM).

Is it necessary to use DMSO in serum sample for studying exosomes?

DMSO is a cryoprotectant used to preserve the cell membrane from any damage that may be caused by freezing like crystal formation for instance. However, the size of the exosomes and liquid content in it is very small as compared to the cells. Thus, we do not recommend adding DMSO while freezing your serum samples at -80°C.

How do you determine the protein concentration in Exosomes?

Protein concentration in exosomes can be detected by Bradford or BCA methods after lysis of the exosomes. This will give you amount of protein concentration in exosomes in µg/µl.