I've had good results with Exoquick-TC - its designed for large volume purification. I would recommend filtering your media with a 0.2micron filter prior to using the kit and that will help reduce contamination with larger vesicles. I've never worked with RAW, but a T75 flask of THP-1 cells yields enough exosomes for several westerns. Hope that helps. Good luck!

We use Exoquick to isolate exosomes from the media of adult cardiac myocytes and from the media of C2C12 cells. We prefilter our media to concentrate it and remove debris (see recent publication Z.A.Malik et al. in Am. J. Physiol) for more details. On each prep we do particle sizing and are getting good results with peak size around 100 nm.

I think all are good but I prefer Exoquick (System Bioscience) since the system and methodology is already set and made to isolate Exosomes. As you know normally due to time and money we don't make new methodologies on every few month and also it's not our priority. My western and protein content in exosomes is working fine with this kit, no problem so far. Only they changed the name and made two kits from one now, one for Cells and another for serum/urine.etc etc.

I think 50 ml SN is enough to have plenty of exosomes to analyze different proteins on the western blot. I hope it will help for the time being?????

We do exosome preps from RAW cells frequently using the classic ultracentrifugation method with PBS wash. Depending on the Ab and marker, we need supernatant from ~ 3 million cells to do Westerns for CD9, CD63, and CD81. Supernatants from ~ 15 million cells is enough for an EM sample.

Hi,

An alternative for pre-enrichment of exosomes is also to use the Totale Exosome Isolation kit. Per-enrichment ca be performed from cell culture media, serum, plasma, urine, and from other body fluids. For increased purity, isolation of potential exosome subpopulations, providing a solid surface for exosomes to dock onto for downstream flow exosomes can be isolated using Dynabeads coated with anti-human CD63 or a more generic approach using Dynabeads coated with Streptavidin in combination with biotinylated prim Abs. Such immunomagnetically isolated exosomes are suitable for downstream e.g. Flow, WB and TEM.

kind regards

Ketil W. Pedersen

We have tried exoquick and did not produce good results at all.....RNA extraction produced horrible results....so we are still on the "classical" UC protocol. However new approaches are on the way, such as gel filtration in sepharose (or similar) that produce apparentely good results .

Forgot to mention that we have not been able to get any of the immunocapture kits or Ab against human tetraspanins (eg, CD9, CD63, CD81) to work for mouse exosomes. The Ab don't cross-react. We've had to screen and build our own panel of Ab.

We tried different isolation kits(exo-quick, exo-pure, etc) for protein content - all have 'contaminates', meaning other proteins found in serum. - like albumin; masking other proteins that are expected to be found in exosomes. So immuno-precipitation with beads is preferable (magnetic beads). for culture media, you don't expect to find contaminating proteins, just keep in mind to use SERUM-FREE media.

I am working on isolation of plasma exosome from pediatric patients and plasma volumn is extremly limited (50-100ul), so traditional UC is not good for the exosome isolation. Our lab ordered one SBI Exoquick kit and one Total Exosome Isolation Kit from Life Techology, but did not generate ANY good results at all !!! waste money. The Exoquick kits did not produce any exosome, but debris under EM, and western blot showed negative bands of two exosomal markers. I dont know where the debris are from. Life Tech kit gave very low yield of exosome, very very few particles under EM. I then tried a trail sample (PureEXO) from 101bio (anybody heard this company before?). EM shows that the particle size ranges from 30 to 250nm, which is good, and i need to test them for Western blot and RNA analysis. update later.

Hi Yu Zhou, I was wondering about your results with your the PureExo. How did it go?

Thanks!

Mattia

Mattia, here is the update: I isolated circulating exosome from 100ul patient plasma using PureEXO and used 2/3 of the exosome for RNA extraction. I have total 12 individual samples, exosomal RNA yeild ranges from 70ng to 110 ng per sample measued by nanodrops. You may argue that nanodrop is not accurate for small RNAs carried by exosomes such as microRNAs. So, I run a RT-PCR of two miRs as a test for quality control of the extracted exosomal RNAs. The PCR cycle numbers are around 31 and 33, which is excellent, I think, because I will run microRNA array next. I also saved same exosome for EM scanning. Clearly, I can see the double membrane encapulated particles with diameter from 20 to 150 nm. I don't know how to attach a EM image to share with you. The partciles are clean and easy to identify under EM, no debris at all. And I am thrilled to notice some patient samples show size difference and stucture changes in their exosomes. en, it inspires me new ideas. :)

Let me know if you need more information. thanks.

Hi Yu,

thanks a lot for your reply! I am happy it is working for you. Rather than microRNAs I will be extracting proteins from serum exosomes. I am really indecided betwewen the exoquick, the one from invitrogen and the PureEXO. Good luck with your work and I'll keep you updated on how it goes on my side!

All the best,

Mattia

Here is TEM image of the isolated exosomes.

Mattia, I performed western bloting of exosomal protein as well using exosomal markers flotilin-1 and CD63. For mouse serum, I suggest to use CD9. good luck with your research on exosomes.

We have tried ExoQuick and PureExo and have had good results with ExoQuick and unfortunately could not get any results from the PureExo reagents. We have had good luck with ultracentrifugation after sucrose gradients which is what we use for all of our cell culture purifications and use ExoQuick for our clinical samples. From Culture the gradient technique has been successful in culture medium taken from 12-48 hr cultures and in volumes of 1ml to 30ml (we simply change the ultracentrifuge and rotors to accommodate). In our clinical samples we have had good luck with the ExoQuick from volumes as low as 100 micro liters of both serum and plasma preps. We have had very poor luck with urine using any of these methods. Best of luck.

I've tried the exosome isolation kit from Life Technologies and had really poor results even when I massively scaled up the number of exosome-secreting cells cultured. I would not recommend it. We use immunocapture with Dynabeads and ultracentifugation and have good success with both.

Hi

Mattia Calissano , did you worked on your project ? if u did, what did you used for exosome isolation? and how its work?

because i am going to extract serum exosome and analysis their miRNA and i am wondering if these kits works or not ?

please inform me

Hi Malak, I have been trying several kits: invitrogen, p101bio and exoquick but without much success from all of them. As I mentioned above, I need to purify exosomes from serum to a level pure enough to extract their protein content and do a mass-spec analysis. Unfortunately all my preparations are massively contaminated with albumin and other serum proteins. Also, I ran a western blot analysis with a good anti-alix antibody but, despite having a signal with a positive control, I cannot see any alix band in my preparations.

My personal idea is that all these kits use a variation of some basic biochemical protocols implemented in the 50's 60's to isolate lipids from blood. They are good enough if your downstream application is a PCR (mRNA or microRNA) as you just need few molecules for that, but definitely not for proteomic analysis especially if your starting material is in the range of 100-300 microliters. Let me or other users here know if you find some solutions!

@Mattia Calissano: It might help if you treat the isolated with Proteinase K as in this paper:

http://www.cell.com/stem-cell-reports/abstract/S2213-6711%2814%2900113-1?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671114001131%3Fshowall%3Dtrue?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671114001131%3Fshowall%3Dtrue

Theoretically it should get rid of all the proteins except the ones inside exosomes.

Hi, I started using ExoQuick-TC in order to isolate exosomes from mouse astrocyte culture media, and I am trying to perform a wb analysis using Alix antibody and characterize the isolated extracellular vescicles as exosomes. It's a starting point of an interesting project, but by now I could not see alix detection on my wb, and I think this could depend on few starting matherial. I hope the kit could isolate exosomes!

Do you know any ELISA kit specific for exosomal markers for mouse?

Looking for solutions!

Suzana- We have found that the Ab used for human exosome isolation and analysis do not cross-react with the murine proteins. Although I don't know of an ELISA kit for mouse exosomes, Biolegend has some fantastic anti-mouse CD63, CD9 and LAMP1 Ab that work for Western blotting.

Try ME-Kit from New England Peptide

I would not recommend ExoQuick or Total Exosome Isolation reagent since these products co-precipitate non-exosomal contaminants and can significantly bias your results. We have recently performed a direct comparison of these commercial kits to classical ultracentrifugation and iodixanol density gradient centrifugation and found the latter to be superior in isolating pure exosomes. Results of eg a microarray are completely different when using ExoQuick or density gradient.

http://www.journalofextracellularvesicles.net/index.php/jev/article/view/24858

I have tried the Invitrogen Total Exosome Isolation from Cell culture supernatant - totally useless! Did not get any qPCR results even from 15ml of supernatant! Now trying Exo-spin..will provide update later.

I have tried Life Technologies Total Exosome Isolation kit from Cell Culture to isolate exosomes and got very good results. I tested the prep using CD63 antibody on a western blot and got a very good signal. Hope this helps.

I've been using the Life Tech reagent for cell culture media as well, and for miRNA qPCR I've had great success (even when the pellet was invisible).  I've scaled this from 1-20 ml of culture medium so far.  Protein has been trickier... not sure if it's the antibodies I'm using (SBI exo Ab kit) or the samples, but even loading 10 ug of protein the bands are difficult to see and the antibodies are not very specific.  Functionally we are seeing interesting changes when adding isolated exosomes to other cell types, but we have concerns about co-precipitated microvesicles visible by DLS so will be testing some additional protocols to try and remove them.  And for those doing RNA isolation, remember that you will likely not be able to quantify by spectrophotometry so try BioAnalyzer if available... you should see a small peak corresponding to miRNA only (no rRNA) in your sample.

Hi, any updates about PureEXO kit?

Do you know if these kits work with other species such as dogs?

Hi, I have used the Total Exosome Isolation from Cell culture supernatant and checked the exosomes with the CD9 ExoFlow beads and had a beautiful single peak flow. I have also tried the Exospin columns and checked with flow and had 2 peaks??? Was able to obtain nice miRNA qPCR on isolated exos from the Total Exo kit. In the process right now of checking with EM. I will update when I get results.

Dear Dr. Yu Zhou and Heather A Hardin, please share your updates. Many thanks.

Thanks everyone, that's very helpful. Quick question, how do you quantify the [exosomes] ? The goal is to inject exosomes in a mice !

Check this out everybody! 

http://www.ncbi.nlm.nih.gov/pubmed/25939587

"Exosomes derived via Total Exosome Isolation Reagent (TEIR) as well as ultracentrifugation (UC) from the SKOV3 cell line were analyzed. Spectra of exosomes derived via TEIR are dominated by a signal characteristic for the TEIR kit that needs to be subtracted for all measurements."

@Heather: getting single beautiful peaks is no sign of lack of artifacts due to the specific isolation method. You can still use a kit, but then one should be careful and honest about the biological claims and conclusions.

@Wassim: One can quantify based on total protein extract and/or NTA. In my opinion protein is better.

That dominant signal is for SERS spectroscopy. I'm looking at CD9 beads coated with exosomes and stained with a FITC marker. Totally different system and analysis. However, you raise a good point. For my negative beads sample I should incubate with some of the precipitating reagent in case there is some interaction with the regent and the beads for background purposes. It is the correct control. Also, that paper had more evidence that the precipitating reagent is better at isolating exosomes than UC and is stated that the reagent has a strong bond for exosomes. Makes me more confident in the reagent. Thanks!

The fact that you are looking at a specific subpopulation (CD9) does not mean that the method of isolation prior to selection is irrelevant. It depends on your downstream analysis and your questions.

The fact that the reagent is sticky, well, if you are using the material for testing its biological interactions and functions then I would argue that you don't want to modify its chemical properties and anything sticky will do that...

As for isolation, if you are not using high centrifugal forces, than yes, it better be sticky...

Total exosome isolation reagents work well, in particular for isolation from cell media. To confirm purity plus count, we routinely perform Nanosight LM10 analysis, Western (CD63, sometimes other markers), qRT-PCR for a panel of exosomal miRNA/mRNA markers, sometimes- EM. 

I have had no luck at all with the TEI reagent, but I work with MDCK cells. It produces a large pellet, but there are no bands for Hsp70, CD63, HLADR, Tsg101, or Alix, even at a 20ug protein load. These are all present in the lysates, though and even appear in typical ultracentrifugation methods at concentrations as low as 3ug. 

Dear Yu,

 which buffer you used to lyse exosomes for western blotting?

Dear Soma,

We have used RIPA buffer for western with success.

Lane et.al. have comported four different kits. If anyone is interested in exosome purification I think this is a very helpful article. Good luck.

Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

Rebecca E. Lane, Darren Korbie, Will Anderson, Ramanathan Vaidyanathan & Matt Trau

Scientific Reports 5, Article number: 7639 (2015)

doi:10.1038/srep07639

You can paste the link to your browser:

http://www.nature.com/articles/srep07639

I would also suggest using Immunocapture based methods for the isolation of exosomes. Other methods including differential centrifugation often comes with problems like contamination of exosomal fraction with aggregates of cellular proteins and debris/particulates. At Novus, we have immunocapture/antibody based kits, immunoplates and immunobeads for the isolation and/or quantification of exosomes. For cultures of RAW or any other mouse cells, I would recommend NBP2-49833 or NBP2-49834. For Mouse biofluids, we have NBP2-49831 and NBP2-49832. http://www.novusbio.com/research-areas/cell-biology/Exosome-research-tools

I agree with Subhash, the centrifugation methods are time consuming and not pure. I also agree with Jan, the precipitation method alone is also impure. The Exospin from Cell Guidance is not as pure as immunobeads, although much better than ultracentrifugation and precipitation methods alone. I use precipitation for the general isolation(cell culture media) then I purify with immunobeads. I love Novus but they just got into exosomes so I haven't tried their products in this line as of yet. I have traditionally used System Biosciences (SBI), they have extensive exosomal products (anything you want), including Exo-Flow, which is a precipitation based isolation and bead based purification kit and can be used with flow cytometry.  

Hi Pablo,

I would not recommend using the commercially available kits, they are easy to use but associated with high levels of contaminant. 

Working with culture media: I have been doing the recommended centrifugation steps to get rid of debris. Concentrate down the culture media using MWCOs to get rid of small protein contaminants. Then run through a SEC gel.

It is important to characterise your elution profiles first and select a good trade off between protein contaminant and EV elution (determined by BCA and TRPS respectively). 

In terms of how much cultured media to run, I use 40ml per harvest. 

We tried all !!!!.

New England Peptide's ME-Kit, a peptide based exosome precipitation, may be a good try, easy and done in 2 hours. We get way more particle from plasma........

In recent study Exo-spin™ had significantly better performance compared to ExoQuick™ especially if we compare the results of the obtained particle purity. However, both kits are unable to resolve the problem with particle heterogeneity (existence of non-exosomal particles).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507751/

We used Exoquick as well as using ultracentrifugation based isolation and then used immunocapture kits against human CD63. Exoquick gives a big pellet before immunocapture as compared to ultra. The Exoquick pellet has a lot of debris as checked with TEM, WB and qPCR for some miRNA. THe ultra based purification is far better. Density gradient based purification leads to more pure exosomes but we loose a lot of exosomes. CD63 immunocapture after ultra gives a very pure exosome population.

Does anybody use PureEXO (101bio)? I'm interested in

Pablo have you then found a good exosomes kit?

THANK YOU

Hi,

I am looking for similar answer . Has anyone found better kit for Exosome isolation from blood plasma (for RNA)? I would like to add that based on literature Total Exosome isolation reagent seems working fine from cell culture media (for RNA) but I cant say so for plasma samples . Article Comparison of isolation methods of exosomes and exosomal RNA...

Has anyone here tried it with plasma ?

Thank You!

I must say that by using precipitation reagents to isolate vesicles, you are getting a sample that contains a lot of protein and lipid debris. Even if proteinase treatment is used one cannot get rid of the abundant proteins in plasma. This undermines all functional or phenotypic studies you do.

In my experience, size exclusion chromatography is much better, although even by using SEC one does not remove all contaminants from plasma!

Thank you for the answer Elina Aleksejeva , I agree with you. But what about UC ? does that also leave exos with lot of contaminants. Can you please share with me your protocol of isolating exosomes by@ SEC. what size columns are you using ?

Neha Rana , You can find the protocol attached to this message.

The protocol is for medium columns (I used it just today to isolate EV-s from 2ml of plasma). There are also smaller and bigger columns depending on your need.

Elina Aleksejeva , Thanks a lot for sending this .I havent heard of the company much though . I will be waiting to hear from you after you characterize these isolated exos :)

Elina Aleksejeva , have you compared recovery using this method versus other isolation methods? (For example, if you know you recover 100ug of protein from 5ml of plasma from method A, how much protein is recovered from the same volume using this column method?) Thanks!

thank you Elina Aleksejeva to share the protocol

Allyson McCune , I have not done extensive comparison of all possible methods myself, however I recall there is plenty of published data. For example, in the following paper they used precipitation, centrifugation and size exclusion in parallel:

Article Precipitation with polyethylene glycol followed by washing a...

Dear Elina Aleksejeva , once you get exosome fractions, can you directly use the entire volume of the EV fraction (either 500 uL or 2 mL if you use the fast SEC method) to extract either RNA or proteins? Or is it advisable to concentrate exosomes before? In which way?

Hi, Pablo.

I recommend kits for small volumes, like when working with patient samples. But if you have a rich resource such as cultured medium, I suggest using ultracentrifugation. You'll need about 220mL of conditioned media from cells grown in exosome-free FBS, but you'll get a very nice pellet of vesicles that will enough for characterization and whatever other analysis you require.

Chiara Carone , for ELISA analysis I do not need to concentrate. In some cases I have used 100K centrifugal filters to concentrate.

There are also RNA extraction kits that can be used on pre-isolated vesicles, but I have no experience with these.

Hi Pablo,

There is a paper comparing all the kits you are mentioning, please find it here:

Article Exosome isolation from distinct biofluids using precipitatio...

The Exo-spin kit seems to be the most suitable when compared to ExoQuick and Invitrogen for CSF, plasma and sera samples.

Let me know if you have any questions :)

Julia

Hey Pablo,

You could also try the Fab-TACS Exosome Isolation Kits. It is a column-based system that targets CD9 or CD81 on the surface of exosomes. It works with reversible reagents and keeps the exosomes intact and functional. You can find more information about the technology here: https://www.iba-lifesciences.com/exosome-isolation.html

The attached application notes show some results you can expect from isolating exosomes from cell culture supernatants using these kits.

Hi Alexandre Helmke, did you get good yield and purity with that kit using small starting material like 750ul of plasma?

Maybe CUSABIO Exosome Isolation Kit is also a good choice. You can find more details (including the operation video) at https://www.cusabio.com/Exosome-Isolation-Kit.html

Hi

Which kits is better for isolating exsosome and exosomal RNA from blood serum !!

Which good! If i take one kit for isolating both or taking kit for isolate each one!

1)COSABIO Exosome isolating kit

2) EVeryRNA™ EV RNA Purification System with SmartSEC™ Single

3) EVeryRNA™ EV RNA Purification System with ExoQuick® EV Isolation

4) Exosome immunocapture and RNA Extraction kit

Have any one tray one of them !!

“Your adivice is very appreciated “

Has anyone tried the MagCaptureTM Exosome Isolation Kit (FUJI FILM) which targets PS. Theoretically it should work well for cell culture across species.

Hello everyone. I have isolated miRNA from exosome. The 260/280 ratio was good but the 260/230 ratio was very bad (0.03). This is the fourth time, i got the same result. Here, I have isolated exosome using differential ultracentrifugation method. What should I do?

NOTE: I have checked the expression of U6 snRNA by RT-PCR using those miRNA derived cDNA. CT value was around 27.