incubated with 5 u03bcl 4 u03bcm diameter aldehyde sulfate latex beads interfacial dynamics portland oreg and resuspended into 400 u03bcl pbs containing 2 fcs exosomes coated beads 20 u03bcl were incubated with the following antibodies anti rab5 santa cruz anti cd63 fitc pharmigen anti cd81 pe pharmingen anti cavelolin 1 clone n 20 santa cruz for 30 minutes at 4 u00b0 c followed when needed by incubation with pe or fitc conjugated secondary antibody and analyzed on a facscalibur flow cytometer bd biosciences purified exosomes were lysed in lysis buffer containing 1 triton x 100 0 1 sds 0 1 m tris hcl ph 7 and protease inhibitors 10 u03bcg ml aprotinin 10 u03bcg ml leupeptin and 2 mm phenylmethylsulfonyl fluoride sigma exosome protein concentration was determined by bradford microassay method bio rad laboratories hercules calif a total of 50 u03bcg of proteins were resuspended in sds sample buffer boiled for 5 min separated on 10 sds page gel and electroblotted on nitrocellulose protran ba85 schleicher and schuell membranes were blotted with antibodies to cd63 diluted 1 50 and rab 5b diluted 1 50 incubated with appropriate hrp conjugated secondary antibodies amersham pharmacia and visualised by enhanced chemiluminescence ecl pierce the novel elisa test exotest according to this disclosure is based on the presence of specific proteins in the exosomes these proteins are shared with cytoplasmic organelles such as endosomes and lysosomes rab 5 and cd63 respectively whose membranes are not shed or recycled as for plasma membrane structures thus excluding the possible presence of structures deriving from membrane disruption ninety six well plates nunc milan italy were coated with polyclonal anti rab 5b antibody clone a 20.