Hi Kathrin,

We also got a stained pellet in our PBS sample when we used ultracentrifugation to wash our exosomes after staining, so we are instead using Vivaspin filters, so the exosomes will stay in the filter and the unbound dye will go through. Then when we add our PBS Control to the cell we do not have any or very little staining suggesting that unbound dye can be washed away with this protocol. We have described it in one of our publications from 2011.

http://www.translational-medicine.com/content/9/1/9

Up side down!!!

Try to add u r staining reaction (unbound dye+exosomes) to the cell pellet and re-suspend and centrifuge to remove the cells and collect the supernatant.

(You might loose some exosome but not all)

goodluck!

Thanks for your answer! I am not sure if I understand correctly. You recommend adding dye + exosomes to the cell pellet in order to get rid of excess dye? And then the supernatant contains the labeled exos?

What I would like to do is adding labeled exosomes to cells to confirm the uptake. But I don't know how to make sure that there are no bound exosomes left on the cell surface.

Thanks again.

You got it right!

I guess if u do it on ice, might prevent loosing the labelled exosomes!

Other ways can be doing a serial dilution of the exosomes ( By limiting the dye) and look for the saturated signal in the uptake experiment.

Did you find any article for the PKH dye and conc of exosomes?

Instead of cells you can also use the exosome coated latex beads (same exosomes) Later you can use those bead for flowcytometry or Lyse to get the total protein for western blot.

1. Try to centrifuge your PKH dyes before adding to exosome / control PBS staining. There might bit PHK deye aggregates already present in the stock solution. Just speculative!

2. I´m not sure whether it might help but in this paper the authors stained exosomes and resident cells with two different colors. Therefore, it is easier to detect exosomes attached to the cell membrane and internalized exosomes. Using FACS analysis a double positive population can then be identified (pre-centrifugation might membrane bound non-internalized exosomes?!)

Good luck!

http://www.ncbi.nlm.nih.gov/pubmed/24335232

Thank you very much Abdul-Aleem and Lucian! I will try what you suggested.

Hi Kathrin,

We also got a stained pellet in our PBS sample when we used ultracentrifugation to wash our exosomes after staining, so we are instead using Vivaspin filters, so the exosomes will stay in the filter and the unbound dye will go through. Then when we add our PBS Control to the cell we do not have any or very little staining suggesting that unbound dye can be washed away with this protocol. We have described it in one of our publications from 2011.

http://www.translational-medicine.com/content/9/1/9

Thank you very much, Cecilia! This is very helpful.

Just one question, why are you washing the stained exosomes at the end with IMDM? Can this be done by any other medium, too?

We thought about dialyzing the stained exos against a large volume of PBS in order to get rid of excess dye, do you think that works the same way?

Thanks again for your contribution.

The reason for using IMDM is that that is the medium of the recipient cells that we used in this particular experiment. So you could use any type of liquid or medium of choice.

Regarding the dialyzing protocol I do not dare to speculate as I have no experience of that.

Good luck and just let me know if you have any questions about the protocol.

Hi Kishore,

The labelled vesicles stay above the filter and can be collected after the washes and the unlabelled dye will go trough the filter and can be discarded. Of course you can add some extra liquid in the end and collect to make sure that the vesicles are not adhered to the filter.

300,000 MWCO is't it too big? as your BSA is about 66000 Daltons only?

Have you tried less thats 300,000MWCO filters.

kishore you should read about the filters they are special they can hold the volume. They are V shaped filters.

Kishore as Abdul-Aleem said, this is not a flat filter, its used to concentrate samples. http://www.sartorius.com/en/product-family/product-family-detail/m-vivaspin-6/?no_cache=1

Abdul-Aleem, one problem you can get with filters with lower kDa cut off is that they clog and no liquid is passing through. So for us these filters have worked the best.

Thanks Cecilia

One more Ques to Cecilia:

I just wonder is there any best filter based method you will recommend for the isolation of exosomes, I believe we all are destroying the exosomes by ultracentrifugations.

Abdul-Aleem, I haven't tried any filter based isolation methods so unfortunately I cant recommend one. We regularly use ultracentrifugation for isolation of vesicles and it works well in our hands.

Hi Kishore,

The time was very dependent on the samples so I usually started with 1.5 or 2 minutes to see how much of the fluid that went trough and then kept repeatedly on centrifuging them for 2-8 minutes till only little fluid was left, but always making sure that the filter did not go dry. So I would not start with 10 minutes because its a risk that all fluid is going through and you damage your vesicles. Then its better to do 3 times 3 minutes to be able to monitor your samples.

Hi Kishore,

We have never frozen our labelled exosomes, but always used them fresh. So I do not know how they would be affected by this. My suggestion is that you labelled them the same day that you are going to use them or in worse case label them the evening before and keep in 4 degrees over night and use them in the morning. If you going to freeze them I think you have to evaluate that first.

Best regards

Cecilia

Hi Kishore,

Sorry for my slow replay. Yes I have tried to refrigerate my sample over night and it worked well.

I hope its going well with your experiments.

Best regards

Cecilia

Hi everyone, a related question- Is this the Pkh dye kit that everyone has been using for exo labeling? http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/mini26bul.pdf. I am planning to order a mini kit as my experiments are in vitro. The amount I need would depend on the concentration of the dye required to label efficiently. If I were to label 500 microgram of exosomes in total for all my experiments, would the mini kit suffice? Any suggestions? 

If you put dye in PBS it will form micelles due to the physiological salts (page 3 product info sheet). Depending on size, these could pellet during ultracentrifugation. Your control should be diluent C and dye, with no exosomes.

HI Cecilia,

Do you check the purity of isolated exosomes and what method you use?

Here we all are discussing staining protocol for isolated exosomes. I want to stain exosomes in conditioned media, is there any specific staining only for exosomes or microvesicles, which will stain only these not other membranes, RNA etc

Hi Meenu,

Well purity is a very good question, that I think that the whole EV community is dealing with right now. At this point I would say there is no good assay. Some try to do ratio between RNA/protein or lipid/protein to try to detect increase contamination of soluble proteins. Some run WB for several (10-20) proteins, both exosome markers that should be present and markers from for example Golgi, ER, nucleus that should not be present. Negative stain in electron microscopy or Cryo-TEM could be used to see if you have a lot of debris or if it is mostly vesicles, but this is not really quantitative or high throughput.

Regarding a stain specific for exosomes there is none. PKH67 is a membrane dye and will label all membranes, which also means that if you do not wash it away it will stain the cell membrane as well. You could construct cells that express CD63-GFP in that way or exosomes are labelled and no washing steps are needed.

Best regards

Cecilia   

 Hi Cecilia,

I am trying to label exosomes with DiI and facing the same problem. Briefly, 100 ng of Exosomes was incubated with 0.6uM DiI for 1 h in the dark at room temperature

with gentle agitation. Do you have any suggestions regarding the protocol? Do you think Vivaspin columns will also work for DiI?

Hi Bhagyashree,

I have no experience with Dil, but Im assuming it would work. However, 100 ng of exosomes sounds like very little material and I think that it will be hard for you to retain that from the filters. We are usually using µg when we are labelling our exosomes. Recently we have also start using optiprep gradients after labelling exosomes and it works very well in removing unbound dye, but also here you need more starting material then ng.

Best regards

Cecilia

Hi Cecelia,

Thanks a lot for your answer. I am sorry I was not clear earlier. But, I do use at least 50 ug of exosomes in the proportion mentioned in my earlier comment (100 ng: 0.6 uM DiI). I can definitely use Optiprep gradients. Could you please guide me though their use?Thanks. 

Hi. I have been trying different methods to stain my exosomes with PKH67 from Sigma. So far I was able to get a yellow pellet after the last washing step after I added the dye but when I quantify the protein concentration it is much lower (about 70 %) than with what I started (around 30 µg). I tried using the vivaspin filters with MWKO 300.000 Da with little success. In fact the filter turned yellow while the flow through was transparent. The remaining solution that stayed above the filter did not contain anymore protein. I quantify the protein using a bratford protein assay. I isolate exosomes via ultracentrifugation (1x 70 min at 110.000 G and 1x 70 min at 100.000 G as a washing step). I added 4 µL of PKH67 Dye solution to 1 mL of Diluent C, stop the reaction with 1 % BSA and either centrifuge 2x at 110.000 G for 70 min or use the vivaspin filters with 300.000 Da MWKO according Cecilia Lässer's suggestions.

Are you doing the PKH labeling at room temperature or on ice? Is 300.000 Da to large for the filters? I added 4 µL of PKH67 Dye solution to 1 mL of Diluent C

 hi Alexander Waclawiczek：

     have you  labelled exosome with pkh successfully?recently, i will label exosome with pkh also？can you send me your protocol?thank you

how much pkh should  i use to label 100ug exosome?

Hi Antonie, 

I am pretty sure that the volume of the exosomes and their amount matters a lot.  When I added an increasing amount of PKH-labelled exosomes to my target cells I saw of course an increasing number of PKH-positive cells in FACS analysis. However, the amount of exosomes needed is probably strongly dependent on the target cells, their 'susceptibility' to exosomes and the exosomes itself (source, purity, ...). Unfortunately most of the publications that use such PKH dyes only poorly describe how they did it and usually just write that the staining was performed 'according to the manufacturer's protocol'. Since the original protocol is for cells and not for vesicles, it remains completely unclear how the stainings were performed.... So far I have always used a small volume of exosomes (e.g. exosomes purified from 200 ml cell culture supernatant by density gradient centrifugation and concentrated to a final volume of 100-200 µl).

I my opinion we have to think about what we need this exosome labelling for and what we want to show and learn from that. In my case I just want to show that my exosomes interact with my target cells and I did that with PKH-labelled exosomes and FACS analysis and microscopy. However it does not show me if my exos just bind to the cells or if there is an uptake, etc. So I guess the results of such staining have to be interpreted very carefully. 

Best wishes, Kathrin

Unfortunately we can not exactly quantifiy our particles because we don't have access to a NanoSight or any similar machine. We might get one in the future...

So far I haven't really quantified the labelled exosomes for this experiment because I just wanted to show that there is an interaction with the target cells. Usually we are measuring the protein content of our samples, in order to have at least a constant reference in different experiments. Nevertheless I know that this is not a good method for exo quantification because the protein concentration probably doesn't reflect the particle amount. Tricky!

How are you characterizing your samples, do you have a particle quantification machine? 

Hi Antonie,

You can use the Bradford Assay and measure protein concentration directly from PBS without lysing the particles.

Yes, I am also using Bradford and intact vesicles. However the protein amount of course strongly depends on the isolation method. With ultracentrifugation and optiprep gradient the protein concentration of my vesicles is always very low. 

Hi Alexander and Kathrin,

Why not lyse particles for assessment of exosomal protein cargo when conducting protein quantification?

Do any of you look at your stained exosomes in a flourescence microscope after removing excess dye from the samples? I always see a lot of background colour and I have tried both gel filtration, ultracentrifugation, and spin filters. I haven't been able to find anyone that actually checked if their excess dye-removal actually worked and in my opinion this is quite critical.

Hi Maria,

I save the supernatants after each washing step and subsequently check the fluorescence (exc. 490nm, emm. 502nm) of each supernatant against my final sample of labeled exosomes and find that nearly all excess dye is lost after 1st wash and any remaining is lost in 2nd wash step.

Hi Phillip,

thanks for your answer. How do you wash the excess dye away? I also check the flourescence of wash as well as labelled exosomes, but as I said - I haven't manage to remove all of the excess dye even though it looks like it from flourescence measurements. The background is always very colourfull in the microscope.

HI

I labeled the exosome with PKH-67 dye and I used ultracentrifugation method for collection and washing after staining. I used nanodrop  (280 nm) to quantify the intact exosomes before and after staining. And I lost 90% of the input. Do anybody else also see a loss like this after staining?

Hi Maria,

Excess dye is washed with complete medium (exosome-free of course) and ultracentrifugation. I read the supernatants and samples fluorescence signal on a plate reader, and there is an obvious difference in the labeled exosome sample on fluorescence microscopy compared to a no exosome control.

Hi Phillip,

How many g's do you ultracentrifuge at and for how long time? I assume you use the PHK-dye :-) I also see an obvious difference on the plate reader and in the microscope when comparing samples where excess dye is washed away and when it is not. But in my opinion, the risk of getting a fake positive signal when applying labelled exosomes in buffer to my cells with unbound dye to my cells is too high. I think that cells will take up the excess dye if it is free in the buffer.

Hi Maria,

We ultracentrifuge at 100,000xg for 70min reach round, and yes I do use PKH dye (PKH67 to be exact). I do not see any reason why there should be unbound dye present in the labeled exosome sample if you wash them two or three times after the initial labeling reaction.

Hi Phillip,

just curious as I haven't tried it myself, but have you tried to ultracentrifuge dye and buffer without exosomes to see if it pellets?

Hi Maria,

Yes, my "no-exosome" control has no pellet and no fluorescence when conducted in parallel to the exosome labeled sample. The labeled exosome pellet should look yellow.

We are dealing with the same problem and we use a so called medium control, exactly like Kathrin Gärtner, prepare in parallel a sample with no exosome and one with the exosome, perform the staining, then quench both with FCS (exosome depleted ! ) and use both for the uptake. In order to have information also about the surface bound exsome, we also have some wells containing exosome at 4° for the duration of the uptake, so we can back calculate the amount of internalized exosome.

Seems were are all having similar problems :-)

I agree that free dye left in the exosome sample will stain the target cells and might lead to wrong results. I am using routinely ultracentrifugation in combination with iodixanol density gradient centrifugation for the isolation of my exosomes from cell culture supernatants. Therefore I decided to do the PKH staining after the first ultracentriguation step (100.000 x g) and loaded these PKH-stained vesicles on the iodixanol gradient. I think this is a good way to get rid of unbound dye and all my controls looked fine. The vesicles will accumulate according to their buoyant density, I pooled the vesicle-containing fractions, washed them again in a large volume of PBS at 100.000 x g to receive my final PKH-stained exosome preparation. 

Hi any one, I am using PKH 26 (Sigma) for labeling cell in vitro, All the cells get stained when viewed under floresence microscope but when i do cuture, after 2 hrs they do not show flouresence, can any body suggest me what is wrong????

Hi Cecilia,

what membrane material you have for the Vivaspin filter? We have similar filter with cellulose, PKH 26 binds to the membrane, they dont pass through.

Thanks!

Anna

Hello everyone,

In relation to this topic, I want to label my exosomes (already purified and resuspended in PBS) with a cell mask dye, just to check for uptake by cells. I quantify my exosomes using Q-nano so I have a concentration in particles/ml. My question is how much dye should I add? Should I quantify by a BCA first ? 

Thank you all

Kristina 

Hi Kristina, 

The question how much dye you should add is very difficult to answer I guess... I think an excess of dye is fine as long as you make sure that you remove all unbound dye thoroughly. 

As I wrote already above labelling of EVs prior to density gradient centrifugation works very well in our hands and separates the vesicles from free dye. However it is recommended to include a control sample without EVs (just buffer and dye), just to make sure that the free dye accumulates in different fractions of the gradient.

Does this help?

Best wishes, Kathrin

We have actually came across another problem concerning labelled EVs. 

In several publications you find experiments which are investigating the transfer of EVs from one cell type to another in a transwell system. Here the EV producer cells are labelled with PKH and transfer of the dye is monitored.

We have tried that as well and purified EVs from PKH-labelled cells. However, we could not detect any fluorescence in the EVs and I am wondering whether EVs secreted by PKH-labelled cells are really PKH-positive.

Considering the biogenesis of different EV subtypes, I expect that vesicles budding from the plasma membrane (aka microvesicles) are PKH-positive, because the dye is present in the membrane. But I guess that exosomes derived from multivesicular bodies do probably not necessarily encounter the dye?

Does anybody have experience with these kind of experiments and can comment on these thoughts?

Thank you very much, Kathrin

 Dera Lasser

could you please tell me your protocol for exosome labeling by pkh dye?

Thanks

Hi, Chongxia

Is the membrance material PKH26 and PKH67 dye the same for exosome labeling?

I saw some researchers use PKH67 for (platelet) microparticle in journal articles.

The membrane dyes PKH26, PKH67, and CellVue Claret can be used to label exosomes as well since exosomes are composed of a lipid membrane.  However, some modifications need to be made to the original protocol provided by Sigma since that protocol was meant for cell labeling.  Essentially, you just need to adjust the dye concentration to apply to the exosomes since the concentration suggested in the protocol for cells won't apply to exosomes (smaller surface area than cells).  Please follow the link here to an article that describes a good starting concentration for use of the PKH dyes. See their Methods section.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072912/pdf/JEV-3-23784.pdf 

I have used both PKH26 and CellVue to successfully label my exosomes in the past.  Please note that you can lose some exosomes during this labeling process so it is a good idea to measure your starting exosome levels (I did this by measuring exosomal protein via Micro BCA assay) then the ending exosome levels to assess loss and determine dosing for functional studies.  (You can also measure exosomes via particle number through other methods like the Exocet Quantitation Kit System from SBI.  A particle count would likely be a more accurate assessment of exosome levels in the sample, but a measure of protein level can still tell you if there is loss during labeling.)  To limit exosomal loss, you can keep the exosome sample in the same ultracentrifugation tube during the wash steps even though the Sigma PKH/CellVue labeling method suggests transferring the sample to different tubes to minimize excess dye presence in the pellet.  It is important to minimize exosomal loss, and I found that this helps.  You will also be getting rid of the excess dye when you quench the sample with BSA (during one of the steps), so excess dye shouldn't be an issue.  I hope this helps!  Please feel free to write me back with questions.  I've been working through labeling exosomes with these dyes for several months now and the method has been working well.  Good luck :)

Hi Megan,

I am new to exosome isolation , but have been successful in just isolation as yet. now I am trying to label with PKH26. Have you done the ultrafiltering step uisng 10kDa filters? Is it to get rid of the unlabeled particles or staining? Is there a difference between unlabeled and labeled exosomes in size, purity, protein content and functionally?

Many thanks,

Chamini