Cell death, membrane depolarization, and membrane fluidity quantification assays

As vesicle production can be associated with extensive cell lysis, cell death was quantified by measuring the fluorescent signal in the cell population using propidium iodide (Sigma-Aldrich, P4864), a membrane-impermeable nucleic acid stain commonly used as a cell death marker. Specifically, red fluorescence is indicative of cellular loss of membrane impermeability. On the other hand, membrane depolarization was quantified by the emission of a green fluorescence signal using voltage-dependent dye DIBAC4(3) (Thermo Fisher Scientific B438). Microscopy snapshots were taken to visualize the population of bacteria under these various dye treatments. Membrane fluidity was assessed by using the Membrane Fluidity Kit (Abcam ab189819), which measures the changes in fluorescence spectral properties of lipid analog probes added to the cell culture. Fluorescence shifts (400 to 470 nm) resulting in changes in membrane viscosity were read in a TECAN microplate reader (Infinite M200 PRO). The ratio of emission at 470 nm to emission at 400 nm was normalized to that of unlabeled cell conditions. In all assays, total fluorescence was normalized to biomass (optical density at 600 nm) in each sample.