title: Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.
abstract: The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.
authors.forename: J Derk
authors.initials: JD
authors.lastname: Te Winkel
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.
authors.forename: Declan A
authors.initials: DA
authors.lastname: Gray
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Seistrup
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam Amsterdam, Netherlands.
authors.forename: Leendert W
authors.initials: LW
authors.lastname: Hamoen
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.
authors.forename: Henrik
authors.initials: H
authors.lastname: Strahl
keywords: antimicrobial; depolarization; membrane permeability; membrane potential; potentiometric dye; voltage-sensitive dye