Thank you for the reply. It was very helpful.

We struggled with that same issue too!

I think it is tough to go by protein concentration since the purity of the exosomes can be quite variable depending on the isolation method. Albumin contamination can be quite substantial depending on the protocol that you use to clean up the crude vesicles. If you do gradients, you should be fine. If you wash with PBS, there can still be a lot of albumin carryover that will throw off the results from the protein assay. I never recommend working with crude vesicles (no wash or gradient). Even when using pre-spun exosome production media for our experiments, a ton of albumin will pellet in the 100,000xg spin.

We resuspend our samples by cell equivalents. For several cell lines we find that a final resuspension ratio of 1 million cell equivalents per uL is a reasonable guideline.

We usually resuspend our exosome pellet in 100ul of PBS/40ml culture media and serves well for our purpose.

Hi Noe,

I think the essential think is to make sure you follow a standardized protocol that allows you to isolate exosomes with a consistant concentration. Basically, I think the key is keeping a certain proportion of the resuspension volume depending on the cell count that you got when you collected the medium for isolation. We resuspend the final pellet in 10uL per each million of cells counted in the flasks (human MSCs). You will have to try different proportions. I know that depending on the type of cells, you may have some more sticky pellet that are very tricky to resuspend.

Good luck

its up to you to decide on the final volume- 50-200 ul is just convenient typical vol. We typically isolate exosomes from 1-10 ml CM, and resuspend in 100 ul PBS