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Langmuir. 2019 Feb 12;35(6):2375-2382. doi: 10.1021/acs.langmuir.8b03635. Epub 2019 Jan 30.

Using Imaging Flow Cytometry to Quantify and Optimize Giant Vesicle Production by Water-in-oil Emulsion Transfer Methods.

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Institute of Biology and Information Science, School of Computer Science and Software Engineering, School of Life Sciences , East China Normal University , Shanghai 200062 , PR China.
Laboratory for Artificial Biology, Centre for Integrative Biology (CIBIO) , University of Trento , 38122 , Trento , Italy.
Chemical and Biological Engineering , University of New Mexico , Albuquerque , New Mexico 87131 , United States.
Department of Molecular Biology , Massachusetts General Hospital , Boston , Massachusetts 02114 , United States.
Center for Computational and Integrative Biology , Massachusetts General Hospital , Boston , Massachusetts 02114 , United States.
Department of Genetics , Harvard Medical School , Boston , Massachusetts 02115 , United States.


Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 μm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here, we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol byproducts, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles, about 10 times or higher than that of vortex and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.

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