Hi David,

We routinely collected exosomes from 20 - 50 mL of conditioned media from SH-SY5Ys (normally 2 x T175 flasks), purifying by ultracentrifugation. This gave us enough sample (50 uL concentrated exosomes) for EM and 1 lane of SDS-PAGE (WB). Different cell lines produce different amounts of exosomes however. We didn't use the commercial kits because they collect a lot of vesicular material along with the exosomes. Be careful if you are using serum in your cell culture as it contains exosomes and it is necessary to remove them by ultracentrifugation prior to adding it to your culture media.

Thanks Adam. 

Quick question, I am a bit afraid of removing my exosomal pellet as it is not visible when I do the Ultracentrifugation. Any advice for that? I am thinking of either leave a little of the supernatant or suck it all but putting the pipette in the side as the pellet should be stick in the bottom of the tube.

Yes it won't normally be visible. Be very gentle when removing the supernatant and leave some in the bottom of the tube, otherwise you will draw up a lot of the exosomes - they can be easily dislodged. Good luck!

Agree with Adam, start from 20-25ml of the conditioned media.

Check the Exiqon and Qiagen sites: both companies have kits for exosome or exosomal RNA extraction from the different biofluids. We use Exiqon kits with the exosome precipitation (instead of ultracentrifugation) to collect up to 1010 exosomes from 0.5ml of the cancer patient's serum. You can wash and resubstitute pellet with precipitated exosomes in any volume or proceed to the small RNA extraction step.

Also, after ultracentrifugation you always have some amount of the supernatant left. This super has its own small RNA and can contaminate your "exosomal RNA sample". By using Agilent Bioanalyzer we showed that this contamination can be quite significant. High level of t-RNAs shows that the source of this contamination - destroyed of died cells.

I wouldn't be worry about the serum exosomes. First, they are of bovine origin as well as small RNA in them. Second, level of the bovine small RNA suppose to be the same in all your cell plates. Hence, even if some of these small RNA will be recognized (by your assay) as a human one, it will be no changes in the expression level and will not affect you final results.

I disagree with the above suggestions.

Exosomes in bovine serum:

When you are producing data and finally making claims where you know the source material could have a baseline contamination (e.g. from bovine exosomes) then I would rather worry about cleaning up that base line. In a sense you are right that it would be in all samples, but this only is half acceptable if you are studying control vs. treatment, while it's really not advisable if you don't have a comparison and you are going to do intrasample quantification or comparisons over time. Besides, even if you are doing a comparison between groups, the dirty baseline will affect your true sensitivity range for detection and ad unwanted noise that you cannot compensate or normalize for. 

If I have speckles on my lens I will count more stars in the sky, but at least for a lens i always have the exact same speckles and can count them away afterwards…

Exosomes isolation kits:

more studies are suggesting that the material obtained by these kits is different than that obtained by UC and that the membranes do modify the vesicles. Im not arguing strongly pro or against one method here, as long as one is aware of these so far little known modifications.

Thanks everyone for the suggestions, I totally agree with Virginia about the serum conditions,it is always better to be as clean as possible. I will give a goal a soon as the UC arrive. Finger crossed =)

Virginia, first of all you always compare. You compare treated vs non-treated samples, you compare different time-points. Small RNA profile from one sample doesn't give you much information. You simply must have a negative and positive controls in your experiment to compare with. Second, as I mentioned the bovine (or horse) small RNAs  would hardly "contaminate" the human miRNA assays. People who develop these assays completely understand the problem of cross-species contamination and minimize it. Third, what do you suggest to do about bovine (or horse) exosomal RNA in the media? How do you expect to clean your samples from the "wrong" small RNA?

About exosome isolation kits: I don't understand what membranes you are talking about. Could you please clarify it for me?

Normally (also found in literature) people first clean the FBS with several hours at 100000xg.

Virginia, I completely respect your opinion, but "normally" means "almost all" which is not true.  miRNA labs I know don't do it. They just ignore the FBS exosomes (see my previous post). Also, we still don't know how important these exosomes for the in vitro cell culturing. We need to test how it affects cell growth before we start to use FBS without exosomes.

Please post the references you are talking about (at least PubMed number): its really interesting to read papers you mentioned.

Again, what membranes you was talking about? Is it some special kit or some of your techniques? It would be nice to hear some details.

the volume of cell media for exosome isolation will depend on cell type, density, media, timing but also isolation method and of course your downstream assay- how many exosomes or their cargo you need. for ultracentrifugation I'd use 30 ml, for Total exosome isolation reagent- 1 ml. this will allow to recover plenty of exosomes- enough for Nanosight, westerns, qRT-PCR, other types of analysis (but not for RNA sequencing- this will require more)

compared to the commercial kits, we also think that ultracentrifugation is better. We used a kits called 101-bio(USA) and another kit(ribobio,china) which both could get exosome, but the output is very low from 2-4ml medium.besides, all commercial kits are expensive containing lots of PEG.

Hi everybody, I would like to isolate exosomes from 1-3 mls of conditioned cell media and, due to your previously comments, it seems that is not possible by ultracentrifugation. I will appreciate if anyone knows an alternative method to exosome isolation from small volumen of conditioned cell media. Thank you very much in advance.

Gema,

these might help:

http://www.thermofisher.com/us/en/home/life-science/cell-analysis/exosomes/exosomes-webinars.html?cid=lsg_bid_global_sampleprep2017_CP1197_dynabeads_PG1711_exosomes_native_RGnative_researchgate_native

http://www.thermofisher.com/us/en/home/life-science/cell-analysis/exosomes.html?s_kwcid=AL!3652!10!76965845723794!76965867133056&ef_id=VzPP7QAABMfgiUrS:20171130135301:s

http://cshprotocols.cshlp.org/content/2015/4/pdb.top074476.full

Hi everybody

I would need to known how many protein you load in your sds page