. Isolation of microvesicles and exosomes by filtration and estimation of normal reference range in blood plasma

Current protocols for the isolation of microvesicles (MVs) and exosomes, which in the main focus on differential centrifugation, vary considerably. In an attempt to set a new standard, we describe a filtration protocol for isolating phosphatidylserine-positive MVs (larger than 200 nm in diameter) and exosomes. The key preparative step to successfully isolate both MVs and exosomes to a high degree of purity was a gentle sonication to break up exosome clumps. Filtration through a 200 nm pore size Millipore filter allowed for collection of exosomes in the filtrate. The larger MVs could then be recovered from the filter. Annexin V-PE MVs were sized and quantified using Polysciences Polybead Microspheres (200 nm) and BDTrucount tubes, respectively, on a FACS CaliburTM flow cytometer. The normal reference range from normal human donors was found to be 0.512.82105 MVs/ml. Freeze/ thawing of samples had little effect on MV counts and with age MV levels seemed only marginally reduced. Fasting status also affected MV levels, appearing up to 3-fold higher in fasting individuals. Smokers had reduced MV counts and nicotine reduced MV release from THP-1 cells. Funded by: Cellular and Molecular Immunology Research Centre/ Faculty of Life Sciences and Hammersmith Medicines Research. S.K. received a VC scholarship (London metropolitan University).