Handling Purified Exosomes
It is not of concern at the crude isolation stage, but as the degree of purity increases, adhesion to the tube or the pipette chip becomes noticeable. I imagine there are many who have experienced exosomes going missing in the final stage after they reached the density gradient centrifuge stage. Most exosomes with a high purity following a density gradient fractionation disappear when they are condensed using an ultracentrifuge. In other words, they stick to the tube and cannot be resuspended. At the stage of crude purification, I believe various components (serum protein, etc.) that are mixed in large amounts with exosomes coat the tube and prevent exosome adhesion.
I presume all researchers who handle highly pure exosomes have an experience with this adhesive characteristic of exosomes. Aside from ultracentrifugation, regular pipetting and simple operations such as dispensing samples into plastic tubes quickly decrease the number of exosomes, which is a nuisance for researchers.
Because of this we conduct tests on tubes and pipette chips during preparation, and use the ones that are least susceptible to adhesion.
Also, we use ultra-clear tubes that are highly transparent to prioritize pellet collection with the centrifugal tubes. In washing the fraction obtained from density gradient centrifugation, the pellets are at the very bottom when using the swing rotor, and with the angle rotor we take into consideration its angle when making the collection. Adhesion to pipettes is unavoidable in the process, and it is necessary to plan the experiments with this loss in mind. To avoid the loss, if a density gradient medium such as OptiPrep does not influence the proceeding steps, we conduct the experiment without washing. In conducting particle measurement on the unwashed sample using NTA, please do not forget to set the level of viscosity to match the concentration of the medium.
Highly pure exosomes are also very sensitive to frozen storage. There is no noticeable impact at the crude isolation stage, but after fractionating with density gradient centrifugation, freezing would result in losing a percentage of the particles, which is also a source of headaches for researchers.
We use the non-frozen "first squeeze" for the most important experiments, and then break the batch into small portions and use the "second squeeze" that is frozen only once. For experiments that are not influenced by the decrease in the number of particles, we use batches that have been frozen more than twice. For freezing, we use liquid nitrogen and store at -80oC.