title: A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma.
abstract: The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 μm in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 μm pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 μm) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 μM) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects.
authors: Cellular and Molecular Immunology Research Centre, Faculty of Life Sciences, London Metropolitan University, London, UK.
authors: Ryan
authors: R
authors: Grant
authors: Ephraim
authors: E
authors: Ansa-Addo
authors: Dan
authors: D
authors: Stratton
authors: Samuel
authors: S
authors: Antwi-Baffour
authors: Samireh
authors: S
authors: Jorfi
authors: Sharad
authors: S
authors: Kholia
authors: Lizelle
authors: L
authors: Krige
authors: Sigrun
authors: S
authors: Lange
authors: Jameel
authors: J
authors: Inal
mesh_terms: D000328:Adult; D000367:Age Factors; D000368:Aged; D017304:Annexin A5 / Q000378:metabolism; D015703:Antigens, CD / Q000097:blood; D002460:Cell Line; D055252:Cell-Derived Microparticles / Q000378:metabolism / Q000648:ultrastructure*; D055354:Exosomes / Q000378:metabolism / Q000648:ultrastructure*; D005215:Fasting / Q000097:blood; D005260:Female; D005374:Filtration / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D005615:Freezing; D006801:Humans; D008297:Male; D008851:Micropore Filters; D008875:Middle Aged; D010718:Phosphatidylserines / Q000097:blood; D010949:Plasma / Q000166:cytology; D010980:Platelet Membrane Glycoproteins; D012016:Reference Values; D012727:Sex Characteristics; D012907:Smoking / Q000097:blood; D060149:Tetraspanin 30; D055815:Young Adult
chemical_list: D017304:Annexin A5; D015703:Antigens, CD; C554586:CD63 protein, human; D010718:Phosphatidylserines; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30