Hi Du, don't worry if pellet is strongly adhered to the tube, it's much better to have a big pellet than an invisible pellet. With regard to how to re-suspending it, use 1ml of 0,2 nm filtered PBS and pipette up and down gently, without touching directly the pellet. It's probable that it forms lather but no matter, just keep pipetting up and down until it is completely dissolved.

Kind regards,

FM

The  problem with your pellet is likely that a there is a large amount of albumin in your media.   Most media are supplemented with FBS or similar products.  For serum derived products albumin is the predominant protein, as it is abundant in the blood. It is also very sticky.  We frequently culture without serum in the media when we plan to  collect exosomes 1 to 2 h after a stimulus.   One way to remove much of the albumin is Affi-gel blue from Biorad.   We have used this approach to remove albumin when isolating exosomes from serum.  You can estimate your albumin content based on what was added to the media.  Serum contains 3.5-5 gm of albumin/100 ml.  The best way to remove albumin  would be to pour the Affi-gel blue into a column and let it settle (a couple of minutes). Then  add your media with exosomes  collect the flow through followed by one wash of equal volume  of PBS.  The albumin sticks to the  Affi-gel beads in the column, and the large exosomes flow through.   Alternatively,  you can add  the media to a slurry of Affi-gel blue in PBS incubate gently shaking for 10 minutes and then pour into a conical tube(s) and let the Affi-gel blue settle to the bottom.  The Affi-gel beads are large and heavy and will readily settle.  You can centrifuge at 1000 g to make the pellet tighter and improve exosome yield.  Take supernatant  and ultracentrifuge as you have been doing. Final pellet will be smaller and cleaner.  If this doesn't solve problem,  you are either not using enough Affi-gel blue(which can be regenerated to use again) to remove the albumin, or you will also need to remove the gamma globulins (antibody content of serum) with a similar approach.

Hi, If the exosome pellet is tough to dissolve in pbs you can either let it sit for a few hours at RT or 37c then pipet up and down; or add more buffer and pipet to dissolve; or gently vortex. 

kind regards

ketil

Anne is right on the money with the serum-free media! It is important to replace the media with serum-free media for about 24 hours before you harvest the exosomes. By doing this, you will find the pellet will be less sticky because there will be hardly any serum proteins left to stick to the exosomes (which albumin is known to do!). We have successfully performed our isolations this way with both melanoma and breast cancer cell lines.

add buffer and let it sit for several hours. can gently vortex or pipet. obviously increasing buffer volume helps. Dont sonicate and dont heat beyond 37C

I usually add 100ul PBS supplemented with protease inhibitors and centrifuge the tube at 4000g x 1min placing the pellet in an inverted position, that’s facing the rotor axis. This will make that the centrifuge force detaches the pellet and make it more amenable to pippet up and down and dissolve the pellet afterwards. If it is really a stubborn pellet, 1-5mM EDTA added to the PBS might help.

Hi -

I just began isolating exosomes and was wondering if you guys could tell me what your pellets look like. I generally collect exosomes in serum free media on top of Py2T cells. Today was the first day I spun down a very large volume and the first day I could see a pellet. Mine was relatively large - basically like a cloudy area on the side of the tube and the middle was almost brown. I definitely have seen people finding cloudy pellets but not necessarily the brown center, though I was thinking this may be due to the higher concentration of exosomes at the maximum force spot in the tube. Has anyone seen anything like this? It also didn't seem very sticky, but as per your answers, I may expect that since I am using serum free media.

Thanks in advance!

I doubt that the brown in your pellet relates to exosome concentration if you are extracting exosomes from a cell culture. If it were from blood then I would say it's probably blood cell debris. But in your case, it's more likely that what you are seeing is cell debris that didn't get removed throughout the extraction protocol. Exosome pellets should look white, with a bit of cloudy stuff around it. Colour indicates contamination with other stuff you don't want.

Hi,

I am facing few difficulties during extraction of exosomes.

I use very high volumes of exosome depleted FBS containing conditioned medium to harvest exosomes from breast cancer cell lines. After the 2nd 120000g centrifugation I see a mixture of small white pellet at the bottom of the tube. But as soon as I start dissolving it in PBS, they seem to clump together. No matter how hard I try it remain clumped together. At the end when I try to quantify the protein concentration with BCA method, I tend to get very less protein equivalent amount (way too less than expected).

Is it because the clumped thing are exosomes and I am not dissolving them properly or could it be some other reason?? (I assumed that clump could be cell debris so I picked it out of the tube)

Any advise will be of great help.

Thanks in advance!

It sounds like you removed the exosomes my friend! That white, clumpy pellet is the exosomes. What should be going on in your protocol is after you've spun down the pellet, pipette off as much of the supernatant (excess liquid) as possible. I'm assuming you'll have multiple tubes, so then you want to serially resuspend your pellets in PBS, tube by tube. In order to break up the exosome pellets, you need to pipette them up and down vigorously, with the tip under the surface of the liquid at all times (and not too roughly, because you don't want to bubble the liquid or burst the exosomes). I hope that helps!

Thanks alot for the prompt response. :)

I will try what you've suggested. Actually, the pellet looks a bit like crystals somehow (which made me suspicious about it), like extended white pellet and then they clump together during pipetting.

Do you think sucrose gradient can help to get rid of this clumping phenomenon?

Thanks in advance!

Clumping is normal and desirable. After all, the aim of the 2nd fast and long spin is to aggregate the exosomes into a pellet. And if this is after the second spin at 120,000 x g, then there's very little likelihood that you're looking at contamination. So clumping is not an issue I would be concerned about. The extension of the pellet is perhaps occurring because you're not spinning it for long enough (are you doing at least 90 min?).

Hey

thanks again..

yess I am spinning it for 90 min or 120 min

all the small white pellets appear at the bottom of the tube (SW41 rotor) and form a big giant pellet or clump after pipetting it up and down.

I wish to use intact exosomes for my experiments so I dont want to be too rough to them.

How can I get rid of the clumping phenomenon ? Any experience with that ??

I was thinking of trying sucrose gradient ( to get rid of albumin which could be causing this clumping to occur) or EDTA in my PBS ( as I saw few people mentioned about using it to get rid of clumping).

Do you use intact exosomes for your experimental procedures or exosome lysates ??

It takes a fair bit of force to break open exosomes, so you should be fine with vigorous up and down pipetting of the PBS resuspension as I mentioned. Ask yourself WHY you need to stop the clumping? I've done protocols that require intact and exosome lysate. Clumping is not an issue for either. If it's really bothering you, I guess you could try a sucrose gradient, but you're likely to lose more exosomes that way. So I suggest doing your differential centrifugation protocol (this time, without throwing away the pellet), and doing a western blot to check for the presence of albumin. Then you'll know whether your pellet has too many proteins in it. But get the basic protocol right before thinking about alternative approaches ;)

I wanted to avoid clumping so as to get a measurement of protein amount equivalent of exosomes as I wish to treat my cells with desired amount of protein equivalent of exosomes to get a physiological response. But because of this clumping I am unable to get any considerate amount of protein content in PBS. I will try your suggested ways, I hope it works this time.

Thanks alot for the suggestions.

I also got clumpy pellet (a "huge" pellet beyond I thought) and I need intact exosomes, and I am trying to add some EDTA and put them into 4 C overnight to see what will happen tomorrow morning.

In my experience, having an exosome pellet difficult to dissolve means that the pellet contains other things in addition to exosomes, like proteins or polysacharids. I would recommend to do a sucrose gradient or any other gradient to clean the exosomes.

I have also met this problem. I tried to purify EVs from Hela cells. The 110,00g pellet could be seen very clear and can not be resuspended in PBS no mater what you do. Interestingly, the WB loading buffer is also difficult to resuspend it, suggesting this pellet contains a large amount of non-protein substances.

Did any of you guys finally found a successful way to suspend this pellet in PBS? The vigorous pipetting and incubation at 37 degree do not work in my case.

Thanks in advance,

I have this problem too. The vigorous pipetting and incubation at 37 degrees do not work in my case. Should I try EDTA?