Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells

• Published in: Microbiological research Vol. 200; pp. 25 - 32
• Main Authors: Volgers, Charlotte, Benedikter, Birke J., Grauls, Gert E., Savelkoul, Paul H.M., Stassen, Frank R.M.
• Format: Journal Article
• Language: English
• Published: Germany Elsevier GmbH 01-07-2017
• Subjects: Antibodies; Bacteria - cytology; Bead-based flow-cytometry; Cell Line - cytology; Cell Line - microbiology; Cell Membrane; Colony Count, Microbial; Epitopes; Exosomes; Extracellular vesicles; Flow Cytometry - methods; Humans; Macrophages - cytology; Macrophages - immunology; Macrophages - microbiology; Membrane vesicle quantification; Membrane vesicles; Moraxella (Branhamella) catarrhalis - classification; Outer membrane vesicles; Pseudomonas aeruginosa - cytology; Transport Vesicles - chemistry; Transport Vesicles - immunology; Membrane vesicle quantification; Extracellular vesicles; Membrane vesicles; Outer membrane vesicles; Exosomes; Bead-based flow-cytometry
• ISSN: 0944-5013; 1618-0623
• DOI: 10.1016/j.micres.2017.04.003

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During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30–300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release.