title: Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.
abstract: We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.
authors.affiliation: Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Yamadaoka 1-5, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sunami
authors.forename: Filippo
authors.initials: F
authors.lastname: Caschera
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Morita
authors.forename: Taro
authors.initials: T
authors.lastname: Toyota
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nishimura
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Martin M
authors.initials: MM
authors.lastname: Hanczyc
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D002469:Cell Separation / Q000295:instrumentation*; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes; D005612:Freeze Drying
chemical_list: D005456:Fluorescent Dyes