Hello Yaniv Gil

When the tube is centrifuged for the first time a compact pellet will form at the bottom of the conical part of the tube. During the second centrifugation step with increased RCF in the same tube already having a pellet, may/may not result in the formation of a compact pellet and as a result while separating the supernatant there are chances that the pellet formed during the second centrifugation step with increased RCF may just get mixed with the supernatant.

There could be a probability that the pellets formed in the subsequent centrifugation steps with increased RCF may/may not form compact pellets. So, using the same tube would mean compromising on the quality of the technique.

I hope this helps.

Best.

I agree with Malcolm. Also, changing the tube at each centrifugation step would help you control what you're doing, and verify that your pellet is actually being removed from the supernatant. Otherwise, the risk is that the first big pellet masks the small/fine pellet that you get with the second round of centrifugation.

Hi Yaniv Gil

If you do not change the tubes for exosome isolation then you will end up with a mixture of cells debris and all types of EVs.

For isolation of exosome through ultracentrifugation follow three main steps, in which the first step ( removal of cells debris,first-300g,5min then 2000g,10min,4 deg) need to discard the tubes. For the 2nd step, the high centrifuge (for removal of other extracellular vesicles) and the last step, the collection of exosome has special type of plastic tubes that doesn't need to be discarded but are washed and sterilized after use for the next usage.

Regards,