I am trying to isolate extracellular vesicles from MDA-MB-231 cell conditioned medium. I collect the medium 48 hours after seeding the cells.  I have been seeding about 5 million cells per 150 mm plate in 18 ml extracellular vesicle-depleted, serum-containing medium.  I collect and combine medium from 3-150 mm plates yielding about 54 ml medium.  At the point of collection, I count the cells and I have about 50 million cells in total across the three plates.  Upon collecting the medium, I immediately begin performing a series of centrifugations to clear cells and cell debris, and ultracentrifugations to isolate the vesicles.  I have tried various methods for isolating these vesicles such as the ExoQuick-TC commercially-available reagent from SBI as well as notable centrifugation methods like: Lässer, C., Eldh, M., Lötvall, J. Isolation and Characterization of RNA-Containing Exosomes. J. Vis. Exp. (59), e3037, doi:10.3791/3037(2012) and Clotilde Thery, Aled Clayton, Sebastian Amigorena, and Basic Protocol 1 from Graca Raposo Current Protocols in Cell Biology (2006) 3.22.1-3.22.29.  After various tries, I have never been able to see a pellet at the end of all the ultracentrifugations.  Additionally, after performing exosome quantification using the Exocet Quantitation System from SBI, I found that I am getting very low vesicle yields (levels close to the standard curve blank).  I also went on to isolate protein from 25 ul of vesicles suspended in 1X PBS and performed a BCA protein assay and found that on average I am obtaining only 0.13 ug/ul of vesicle protein or about 3.25 ug total protein in the 25 ul sample.  Is this what others are obtaining?  The trouble is I tried to run a Western blot for known exosome markers, CD9, etc. and since I loaded such a small amount of protein (1.95ug in an effort to save sample), I am not able to detect bands for these markers.  I know that the low protein level is at fault for being unable to detect these markers since I ran a test to determine how much protein I need to load in order to see a band for them.  Based on published literature, breast cancer cells should release higher amounts of extracellular vesicles than healthy mammary cells so its odd that I am not able to isolate more (http://www.ncbi.nlm.nih.gov/pubmed/24462375).  Unfortunately, I do not know of other researchers working on extracellular vesicles at my institution, so I have been struggling with this on my own and I am totally new to this field.  Any suggestions or ideas as to why I am not obtaining higher vesicle yields are greatly appreciated as well as any advice as to what I can do to improve my isolation method.  Please let me know if I missed any critical information and I will be sure to write back.  Thank you in advance!