What was the volume of cell culture media used for the isolation? I am working with cell culture supernatants for exosome isolation, for getting a better yield i scaled up the culture conditions..

Years ago a research fellow and I isolated exosomes from human cord blood CD34+ cells using ultracentrifugation. If I recall correctly, we spun the SN [36 ml] at 100,00 x g, and resuspended the pellet it ~0.5 ml of storage buffer. I don't recall the exact composition of the buffer, but it was not Tris based, phosphate based and had no protein [BSA]. Once prepared, we stored the exosomes on ice in the refrigerator. I did flow cytometry to confirm expression of CD63 and a second antigen - again, I can't recall what it was. I've been retired several years, and much of this is lost.

Ultrafiltration / Vacuum concentrate your sample

Without knowing which cell culture and exosome are the target, it is known that exosome isolation kits are developed aiming to capturing by antibodies raised against the surface proteins of exosomes. It is also well known that isolation kits may miss some types of extracellular vesicles due to the lack of some surface proteins in exosomes. This could be the reason for the low recovery and sample composition may also prevent some affinity binding between antibody and surface targets (considering the cell culture medium is the sample, lysis was not applied and the supernatant was collected). I prefer physical techniques such as ultrafiltration enriched medium subjected to ultracentrifugation by density cushion, or SEC. Exosome isolation kits are a bit error-prone approach depending on the type of cell and exosome to investigate but physical ones are universal to apply.

Good luck