Frequently Asked Questions

Lyophilized 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.

Can we block/inhibit the release of exosomes from cancer cells?

Dimethyl ameloride (DMA), Sphingomyelinase imhibitor (GW4869), and Glucosyl ceramide synthase inhibitor (di threo-1- 1phenyl-2-de canoylamino-3-morpholino-1-propanol) have been shown to inhibit the release of exosomes in different cell lines. Alternatively, shRNA knock down to Rab27 or Rab35 would be the ideal candidates for inhibiting exosome release.

Isolation tools

What are the current exosome Isolation products available from HBM?

We offer a wide portfolio of products specifically for exosome isolation. Our Isolation tools provides solutions for isolating exosomes by multiple techniques, such as immunoaffinity, chemical precipitation, size exclusion chromatography (SEC). For more detailed information, please go to http:// http://exotest.eu/online_orders/exosome-capture-and-isolation.

Could the isolated exosome pellet be visible to naked eyes from human plasma and serum using EXO-PREP Reagent?

Yes, pellet is in general visible from a minimum amount of 100 µl of plasma and 250 µl of serum.

Could the isolated exosome pellet be visible to naked eyes cell media and urine using EXO-PREP Reagent?

Pellet is lightly visible from 1 ml of cell medium or 5 ml of urine.

How to make sure that the exosomes are isolated successfully by your exosome isolation tools?

You can perform analysis for checking the exosome size (NTA). You can do a western blot analysis on the isolated, purified exosomes using antibodies to exosomal markers (CD81, ALIX, TSG101, CD9). You can check your vesicles by electron microscopy (TEM).

Is there any specific marker for exosomes isolated from adipocytes?

It has been shown that almost all adipocyte-derived exosomes express fatty acid binding protein (FABP4).

Why specific isolation of exosomes is important?

If you are interested in selective and specific exosomal marker expressing exosomes, then we recommend immunocapture methods of exosomes isolation i.e. using immunobeads or immunoplates. However, same exosomes can express more than one exosomal markers.

Do you have exosome isolation reagents from Stem Cell Media?

Yes, we do have exosome isolation kit for isolating exosomes from stem cell media.

What are the advantages of HansaBioMed exosome isolation tools as compared to ultracentrifugation?

· Ready to use, easy and fast protocol

· Time and money saving

· We can process more than a few samples at a time

· Small starting sample volume

The data sheet says up to 100 µl of starting sample volume. Do you have any information on how much protein or RNA that yields? Also have you tested with lower or higher sample volumes?

100 µl is the best volume of fluids to load on ELISA plates. Loading 200 µl does not substantially change the vesicle yield, but increases the background. Loading 50 µl is possible, with a consequent reduction of the signal in ELISA assay. About RNA yield: it is possible to isolate RNA from the vesicles captured onto the immunoplates. But, since the vesicle quantity in each well is low, we recommend collecting the vesicles lysates from at least 4 wells to have a consistent RNA yield.

Have you attempted to elute intact exosomes from the ELISA plate?

Although HBM does not provide any tools for eluting exosomes from the ELISA plate, the elution is possible. Customer can try making it with buffers containing a small percentage (less than 0.05 %) of Tween 20. Due to the low yield of vesicles in each well, we recommend collecting together the eluted vesicles for at least 4 wells to proceed with further analyses. We cannot say for sure if this method works. However, using immunobeads, you can purify exosomes and elute from the immunobeads.

How do you isolate and quantify exosomes in one step?

You can use HBM ExoTEST kits for the quantitative and qualitative analysis of exosomes from small amount of human biological fluids (plasma, serum, urine, saliva) or cell media. Transparent and white plates are available depending on the downstream detection approach (colorimetric and luminometric respectively).

What is the minimum volume of starting sample that can be used for exosome isolation?

The data sheet says up to 100 µl of starting sample volume. The quantity of exosomes could vary between samples. Thus, we always recommend a larger starting amount of sample especially for urine and cell supernatant samples. For urine and cell media, we recommend starting with at least 1 ml of the sample volume. Concentrate the urine sample or cell supernatants about 10-20 fold using a spin concentrator. Then load 50-100 µl per well in the ELISA plate. Otherwise, for most of the body fluids the minimum volume tested is 50-100 µl per well. We recommend adding 1X PBS if the volume is less than 100 μl.

How to isolate and purify exosomes from tissue samples?

Since exosomes are secreted, we recommend performing short term tissue explant cultures to isolate exosomes from the tissues. The tissue explants in culture will secrete exosomes. You can collect the cell supernatant and isolate exosomes using the protocol similar to the exosome isolation protocol from cell media.

What are the negative control for exosome isolation?

The below mentioned proteins should be absent from the Exosome preparation http://www.journalofextracellµlarvesicles.net/index.php/jev/article/view/26913 Endoplasmic reticulum; Grp94HSP90B1; calnexin (CANX); Golgi (GM130); Mitochondria (cytochrome CCYC1); Nucleus (histonesHIST*H*); Argonaute/RISC complex (AGO*).

HansaBioMed offers two different size of immunobeads for one kind of sample, 0.4 and 1.0 micron? How to choose the size of the beads to isolate one kind of sample, for example from biofluids?

Yes, we do offer two different sizes of the Immunobeads: 0.4 and 1 micron. The choice of the immunobead size depends on your final analysis. For WB, RNA extraction and profiling, for eluting intact exosomes we recommend the usage of 0,4 um beads.  This is because if you use 0.4 micron beads, the number of beads added per volume is more and the total surface for 0.4 micron is bigger than 1.0 micron. Additionally, they capture better from small volume of samples. 1 micron beads are useful for exosome FACS analysis.

For exosome isolation from cell media (using immunobeads), I noticed use of spin concentrators are recommended to concentrate the cell media samples from 10 ml to 1 ml. Do you have any recommendation for the spin concentrators to use?

You can use the spin concentrators available from any companies with 100K MWCO.

Which antibody you use for the neural exosome isolation plates?

For proprietary reasons, we cannot reveal the name of the antibody used. We have chosen a marker of neuroblastoma well expressed on the vesicle surface that was able to bind the vesicles in ELISA plate and is useful for enriching exosomes from neural origin from those physiologically produced from healthy cells in the body.

Do the exosome isolation reagent (Exo-PREP) precipitate apoptotic bodies?

Apoptotic bodies are much larger (>800 nm), so they will be mostly removed from the sample during pre-spin (along with cells and debris). The exosome reagents are added in the next step, and it precipitates primarily exosomes (30-150 nm).

Exotest / Exofacs

What are other methods of exosome quantification?

Other than ExoTEST and Exo-FACS kits, once can use Electron Microscopy (based on particle size and hence distinguishes between exosomes and other vesicles), Nano Tracking Analysis (optical microscopy based approach to quantify small particles like exosomes) and qPCR analysis for snRNA U6, snoRNA U38B, and snoRNA U43 (quantification based on RNA).

Quantification of exosomes using standard FACS instruments is not possible due to the small size of exosomes: How does your Exo-FACS quantification system works?

After Exosome isolation, the purified exosomes are bound to the 4 µm aldehyde sulfate latex beads (FACS-Beads) followed by the exosome marker characterization via FACS analysis using exosomes specific antibodies.

Exosome characterization using ELISA can be targeted on external protein epitopes on the vesicle. What is the sensitivity of your ELISA assays and how do you recognize the internal proteins?

ExoTESTTM ELISA kits are double sandwich ELISA kits that consists of ELISA plates pre-coated with proprietary exosome.

Quantification of exosomes using standard FACS instruments is not possible due to the small size of exosomes: How does your Exo-FACS quantification system works?

After Exosome isolation, the purified exosomes are bound to the 4 µm aldehyde sulfate latex beads (FACS-Beads) followed by the exosome marker characterization via FACS analysis using exosomes specific antibodies.

Exosome characterization using ELISA can be targeted on external protein epitopes on the vesicle. What is the sensitivity of your ELISA assays and how do you recognize the internal proteins?

ExoTESTTM ELISA kits are double sandwich ELISA kits that consists of ELISA plates pre-coated with proprietary exosome antibodies thereby enabling specific capture of the external epitopes using different biological samples. The detection limit of an ELISA assay is lower than 0.35 µg of overall exosome which is an equivalent of less than 50 pg of targeted exosomes protein. Additionally, detection of external epitopes can be also done by FACS analysis. For internal proteins, we recommend western blot analysis. You can also detect internal proteins via FACS analysis after exosome permeabilization.

For the HansaBioMed FACS kits, CD9 or CD63 antibody is used. Are these FITC conjugated or what is the dye the antibodies are conjugated with?

In Exo-FACS assay kits, antibodies are not conjugated. We provide a secondary antibody Alexa 488.

Can I use heparin or EDTA tube to collect blood sample if I need to isolate exosome from plasma?

No. Heparin will significantly impair the downstream RNA assays, and also the quantification using ExoTEST.

RNA

How do you isolate exosomal RNA?

We offer ExoRNA™ Exosome RNA extraction kits for overall exosome immunocapture and RNA extraction as well as for Tumor-derived exosome immunocapture and RNA extraction.

Can I get enough exosomal RNA from 10 ml cell culture supernatant for qPCR?

Exosome yield depends on the cell confluence, cell type, health of the cells and time of harvest, but in general yes, 10 ml are enough for getting a good yield of RNA for qPCR, by using our Exo-totalRNA isolation kit.

Regarding the lyophilized exosome standards, do you also the characterize their DNA/RNA contents too in addition to the particle number and the protein content?

Lyophilized standards are currently used as a positive control in RNA and DNA kit, so it is possible to use them for isolation of nucleic acids, although each lot has characterized for protein content and particle number (NTA). In addition, many customers are using as mutation control in spike-in experiments.

Can you recommend a miRNA isolation kit for qPCR analysis?

We offer Exo-totalRNA extraction kit. The kit contains immunobeads for overall exosomes capture, all the solutions, columns and elution tubes for RNA extraction. This kit allows high yield of total RNA extraction, including small RNAs, miRNAs.

Why is the exosomal miRNA levels higher in plasma as compared to the serum exosomal miRNA?

Plasma contains a higher exosome amount than serum, since during the serum preparation exosomes can remain entrapped into the clot, thus even exosomal miRNAs are higher in plasma than in serum.

Can you recommend a miRNA isolation kit for qPCR analysis?

We offer Exo-totalRNA extraction kit. The kit contains immunobeads for overall exosomes capture, all the solutions, columns and elution tubes for RNA extraction. This kit allows high yield of total RNA extraction, including small RNAs, miRNAs.

Why is the exosomal miRNA levels higher in plasma as compared to the serum exosomal miRNA?

Plasma contains a higher exosome amount than serum, since during the serum preparation exosomes can remain entrapped into the clot, thus even exosomal miRNAs are higher in plasma than in serum.

How can I normalize mRNA expression in exosomes?

You can use miRNA genes such as miRNA16, miRNA26a, miRNA221, miRNA22, miRNA181a, miRNA181c, miRNA103, miRNA191, let7d and small RNAs (5SrRNA and U6snRNA) that is normally expressed in exosomes.

DNA

How to detect DNA from the Exosome Standards?

Vesicles usually contains a very small amount of DNA, the most of it is fragmented and single stranded, and the quantity is not enough for an agarose gel analysis. DNA can be quantify by nanodrop analysis and by qPCR.  

Is there any possibility of genomic DNA or mitochondrial DNA (mtDNA) contamination using EXO-DNA Extraction Kits?

EXO-DNA Extraction Kits allows the isolation of circulating+extracellular vesicle (EV)-associated DNA and in EVs is contained genomic DNA. It is also possible the presence of some mitochondrial DNA in EVs, but the evidences about this issue are not still very clear. If you want to limit the analysis only to the DNA contained in EVs, it is sufficient to make a DNase treatment after the precipitation of vesicles+circulating DNA, before to add the lysis buffer. Rather, vesicles protect the DNA from the DNase treatment, which eliminates only the circulating DNA fraction.

Is there any possibility of ribosomal RNA contamination using ExoDNA™ Extraction Kits?

Yes, there is a possibility of RNA contamination, since the vesicles contain RNA, in particular miRNAs. Percentage of ribosomal RNA is very small in vesicles. Presence of RNA does not cause problems since it is very quickly degraded. It is possible to make an optional treatment with RNase to eliminate the possible contaminations.

Does EXO-DNAExtraction Kits adopt RNase treatment step?

No, the ExoDNA™ Extraction Kits does not adopt any RNase treatment, but it is possible to do the RNase treatment once the DNA is eluted.

Regarding the lyophilized exosome standards, do you also the characterize their DNA/RNA contents too in addition to the particle number and the protein content?

Lyophilized standards are currently used as a positive control in RNA and DNA kit, so it is possible to use them for isolation of nucleic acids, although each lot has characterized for protein content and particle number (NTA). In addition, many customers are using as mutation control in spike-in experiments.

Antibodies

What biomarker(s) do you use to identify/characterize the isolated exosomes?

As reported in literature (Kowal J. et al. 2016, Greening DW et al. 2016, Lai RC et al. 2016) markers as CD81, TSG101, Adam10, ALIX, flotillin are indicated, in addition to the usual markers CD63 and CD9, for detecting exosomes isolated from cell media and human biofluids.

Is there any specific exosome marker?

Currently, this is an open question, as reported in litterature data. In addition to the usual CD9, CD63 and CD81, ALIX and TSG101 are recognized as specific exosome markers.

Is there any specific marker for apoptotic bodies and exosomes originated from same cells?

Annexin V, thrombospondin, and C3b are typical markers of apoptotic bodies and can be used to differentiate them from exosomes.