Research output: Contribution to conference › Abstract › peer-review
. Isolation of microvesicles and exosomes by filtration and estimation of normal reference range in blood plasma. / Ansa-Addo, Ephraim A.; Stratton, Dan; Jorfi, Samireh; Kholia, Sharad; Krige, Lizelle; Lange, Sigrun; Inal, Jameel.
2012. 14-14 Abstract from International Society for Extracellular Vesicles 1st annual meeting, Gothenburg, Sweden.Research output: Contribution to conference › Abstract › peer-review
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TY - CONF
T1 - . Isolation of microvesicles and exosomes by filtration and estimation of normal reference range in blood plasma
AU - Ansa-Addo, Ephraim A.
AU - Stratton, Dan
AU - Jorfi, Samireh
AU - Kholia, Sharad
AU - Krige, Lizelle
AU - Lange, Sigrun
AU - Inal, Jameel
PY - 2012/4/15
Y1 - 2012/4/15
N2 - Current protocols for the isolation of microvesicles (MVs) andexosomes, which in the main focus on differential centrifugation,vary considerably. In an attempt to set a new standard, we describea filtration protocol for isolating phosphatidylserine-positive MVs(larger than 200 nm in diameter) and exosomes. The keypreparative step to successfully isolate both MVs and exosomesto a high degree of purity was a gentle sonication to break upexosome clumps. Filtration through a 200 nm pore size Milliporefilter allowed for collection of exosomes in the filtrate. The largerMVs could then be recovered from the filter. Annexin V-PE MVswere sized and quantified using Polysciences Polybead Microspheres(200 nm) and BDTrucount tubes, respectively, on a FACSCaliburTM flow cytometer. The normal reference range from normalhuman donors was found to be 0.512.82105 MVs/ml. Freeze/thawing of samples had little effect on MV counts and with age MVlevels seemed only marginally reduced. Fasting status also affectedMV levels, appearing up to 3-fold higher in fasting individuals.Smokers had reduced MV counts and nicotine reduced MV releasefrom 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).
AB - Current protocols for the isolation of microvesicles (MVs) andexosomes, which in the main focus on differential centrifugation,vary considerably. In an attempt to set a new standard, we describea filtration protocol for isolating phosphatidylserine-positive MVs(larger than 200 nm in diameter) and exosomes. The keypreparative step to successfully isolate both MVs and exosomesto a high degree of purity was a gentle sonication to break upexosome clumps. Filtration through a 200 nm pore size Milliporefilter allowed for collection of exosomes in the filtrate. The largerMVs could then be recovered from the filter. Annexin V-PE MVswere sized and quantified using Polysciences Polybead Microspheres(200 nm) and BDTrucount tubes, respectively, on a FACSCaliburTM flow cytometer. The normal reference range from normalhuman donors was found to be 0.512.82105 MVs/ml. Freeze/thawing of samples had little effect on MV counts and with age MVlevels seemed only marginally reduced. Fasting status also affectedMV levels, appearing up to 3-fold higher in fasting individuals.Smokers had reduced MV counts and nicotine reduced MV releasefrom 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).
M3 - Abstract
SP - 14
EP - 14
T2 - International Society for Extracellular Vesicles 1st annual meeting
Y2 - 18 April 2012 through 21 April 2012
ER -