A filtration-based protocol to isolate human Plasma Membrane-derived Vesicles and exosomes from blood plasma

• Published in: Journal of immunological methods Vol. 371; no. 1-2; pp. 143 - 151
• Main Authors: Grant, Ryan, Ansa-Addo, Ephraim, Stratton, Dan, Antwi-Baffour, Samuel, Jorfi, Samireh, Kholia, Sharad, Krige, Lizelle, Lange, Sigrun, Inal, Jameel
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 31.08.2011; Elsevier
• Subjects: Adult; Age Factors; Aged; Annexin A5 - metabolism; Antigens, CD - blood; apoptosis; Biological and medical sciences; blood plasma; Cell Line; Cell-Derived Microparticles - metabolism; Cell-Derived Microparticles - ultrastructure; centrifugation; citrates; Exosomes; Exosomes - metabolism; Exosomes - ultrastructure; fasting; Fasting - blood; Female; filtration; Filtration - methods; flow cytometry; Flow Cytometry - methods; Freezing; Fundamental and applied biological sciences. Psychology; Fundamental immunology; gender; Humans; hydrophilicity; Male; Micropore Filters; Middle Aged; Molecular immunology; monocytes; nicotine; Phosphatidylserines - blood; Plasma - cytology; Plasma Membrane-derived Vesicles; Plasma PMV; Platelet Membrane Glycoproteins; Reference Values; Sex Characteristics; Smoking - blood; Techniques; Tetraspanin 30; Young Adult; Exosomes; Plasma PMV; Plasma Membrane-derived Vesicles; Human; Plasma membrane; Isolate; Immunological method; Blood plasma
• ISSN: 0022-1759; 1872-7905; 1872-7905
• DOI: 10.1016/j.jim.2011.06.024

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 0.2μm 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 0.1μm pore size Hydrophilic Durapore membranes in Swinnex 13mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2μm) 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 (0.51×105–2.82×105 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 50μM) 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. ► We present a filtration method for isolating Plasma Membrane-derived Vesicles and exosomes from blood. ► In a cohort of 57 healthy donors we found the plasma PMV range was 0.51–2.82×105 PMVs/ml. ► Gender and age have no effect on PMV levels whilst fasting increases and smoking decreases levels.