University of Hertfordshire

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).