After ultracentrifugation the pellet will be quite firm, so as long you don't touch it it should not get disturbed. Inverting the tubes will not disturb the pellet, but there will always be some supernatant left behind in the round bit of the tube. There are two ways in which you can eliminate contamination of the pellet with some supernatant.

1) Centrifuge the exosomes through a cushion (for instance 12% sucrose in 50 mM Tris pH7.5). This means you first layer 5 ml of the cushion into your tube, then carefully layer your exosome sample on top. It should have a lower density, so it will not mix if you layer it carefully. The do the ultracentrifugation as before (SW28 swing out rotor). The exosomes will migrate through the cushion and get stripped of all contaminants from the supernatant. When you remove the supernatant, you can use a pipette and suck carefully from the top until youb have just the cushion left. Then invert to remove the cushion, it essentially rinses the sides of the tube and you leave it standing on whatman 3mm paper for 5 minutes, then use a pencil and soft paper tissue to wipe the walls of the tube dry but avoiding to touch the round bottom of the tube. The you can collect your pellet (invisible) in SDS-PAGE sample buffer or whatever you want to use next.

2) Centrifuge without the cushion as you do, invert the tubes to discard supernatant, leave to stand on whatman 3mm paper for 5 minutes, then use a pencil and soft paper tissue to wipe the walls of the tube dry but avoiding to touch the round bottom of the tube, and then wash with 12% sucrose in 50 mM Tris pH7.5, put it in the ultracentrifuge again and repeat the discarding of the supernatant as before. 

Exosomes pellet sticks to the bottom of your tubes. I always pour the supernatants away gently and put tubes on a paper towel upside down and leave them for a few minutes. Then, I try to dry inside the tubes with a tissue paper without disturbing the bottom of tubes, just first 2/3 tubes. 

After ultracentrifugation the pellet will be quite firm, so as long you don't touch it it should not get disturbed. Inverting the tubes will not disturb the pellet, but there will always be some supernatant left behind in the round bit of the tube. There are two ways in which you can eliminate contamination of the pellet with some supernatant.

1) Centrifuge the exosomes through a cushion (for instance 12% sucrose in 50 mM Tris pH7.5). This means you first layer 5 ml of the cushion into your tube, then carefully layer your exosome sample on top. It should have a lower density, so it will not mix if you layer it carefully. The do the ultracentrifugation as before (SW28 swing out rotor). The exosomes will migrate through the cushion and get stripped of all contaminants from the supernatant. When you remove the supernatant, you can use a pipette and suck carefully from the top until youb have just the cushion left. Then invert to remove the cushion, it essentially rinses the sides of the tube and you leave it standing on whatman 3mm paper for 5 minutes, then use a pencil and soft paper tissue to wipe the walls of the tube dry but avoiding to touch the round bottom of the tube. The you can collect your pellet (invisible) in SDS-PAGE sample buffer or whatever you want to use next.

2) Centrifuge without the cushion as you do, invert the tubes to discard supernatant, leave to stand on whatman 3mm paper for 5 minutes, then use a pencil and soft paper tissue to wipe the walls of the tube dry but avoiding to touch the round bottom of the tube, and then wash with 12% sucrose in 50 mM Tris pH7.5, put it in the ultracentrifuge again and repeat the discarding of the supernatant as before. 

Thank you so much for the answers! Cushion is a great idea to remove contamination. If there is some sucrose and Tris left in the exosome prep, does it affect the biological properties of the exosomes? I also wonder what is the minimum volume of PBS or lysate buffer we should use to resuspend the exosome pellet. When I resuspend the pellet in 50-100 ul, there are always a lot of bubbles. I don't know if I got all the exosomes out. My exosome yield is very low. When I increased the centrifugation speed (28,000 rpm for 90 minutes, SW28), I got big pieces of aggregated exosomes that are visible under the microscope. How to dissociate the aggregation?

Pellet could be present even if you don't see it.

Anyway, after an ultracentrifugation, pellet keeps being adherent to the tube for several minutes before it starts re-suspending. Personally, when I have to recovery pellet, i just turn upside the tube emptying the supernatant into a becher, and i keep the tube upturned for about 1 minute. After that, dry the edge of the tube and start suspending the pellet (into 1 ml of PBS or other) to recovery it.

I guarantee you that, if pellet is present, in this way you'll recovery it.

Best regards,

FM

I used to use standard ultracentrifugation technique to isolate microvesicles released from stem cells. With this technique, after the first round of ultracentrifugation, the pellet is rarely visible with the naked eyes. However, sometimes, you might see a very thiny tiny white pellet centered down to the bottom of the ultraclear tube. Anyway, for recovering this pellet, we get consistant yield by just aspirating the supernatant carefully, so that you can almost dry out the pellet. Afterwards, the resuspension of the pellet can be tricky sometimes...because of exosomes agglomerates. Likewise, after the second round of ultracentrifugation, you should get a very thin white pellet into the center of the bottom of the tube. Again, I think your pellet can definitely be difficult to see depending on the initial cells amount which you're working with. In our hands, usually this pellet is strongly adherent to the bottom of the tube. No risk to detach it from the tube when you aspirate the supernatant. You just have to make sure you do not directly aspirate the pellet with your Pasteur pipette. Finally, you could try to stain your exosomes or microvesicles just to track them in order to make them visible so that you can localize the colored-pellet on the bottom of your tube. this is an interesting method to visualize your precious pellet !

Good luck.

Best

You mentioned that you got a low yield of exosomes..Could you please tell me what was  the number of exosomes per cell per hour? I have been having trouble with the yield too.. 

Thanks

Hi Deepa Raghu, I get 30 to 50 microgram of EVs from 25 million cells (counted at the beginning, should expand a lot at the end) over 48 hours. Do you have any idea how to increase the yield?

Antoine, staining the pellet is a very interesting suggestion. I am struggling with the yield as well and I am not sure about the reason. One of my hypothesis is that I can't solubilize the pellet after the ultracentrifugation so staining it could give me an idea, at least, if it is there. How do you stain it? is it a reversible staining method? Thanks!