Membrane Fluidity Measurement by Fluorescence Anisotropy

Fluorescence anisotropy analysis of P. aeruginosa cells were performed as previously described (Fléchard et al., 2018). Briefly, cell pellets of P. aeruginosa H103 untreated (control DMSO 1%) and treated by 100, 50, 25, 12.5, 6.25, 3.12, 1.6 and 0.8 μg mL^–1 of PLFE1 collected after 24 h of incubation with shaking (180 rpm) at 37°C were washed two times (7500 × g, 5 min, 25°C) in 0.01 M MgSO₄ and resuspended in the same wash solution to reach an OD_580nm of 0.1. One μL of a 4 mM of 1,6-diphenyl-1,3,5-hexatriene (DPH) stock solution (Sigma-Aldrich) in tetrahydrofuran was added to 1 mL aliquot of the resuspended cultures and incubated in the dark for 30 min at 37°C to allow the probe to incorporate into the cytoplasmic membrane. Measurement of the fluorescence polarization was performed using the Spark 20M multimode Microplate Reader, equipped with an active temperature regulation system (Te-Cool^TM, Tecan Group Ltd.). Excitation and emission wavelengths were set to 365 and 425 nm, respectively, and the Fluorescence Anisotropy (FA) was calculated according to Lakowicz (2006). Three measurements were performed for each sample and data were recorded using SparkControl^TM software (Version 2.1, Tecan Group Ltd.). The relationship between fluorescence polarization and membrane fluidity is an inverse one, where increasing anisotropy values correspond to a more rigid membrane and vice versa. All values are reported as means of triplicate analyses for each experimental variable.