Membrane fluidity assays with laurdan.

Cells were grown to mid-log phase in LBg (LB with 0.2% glucose). Laurdan (Invitrogen) was added to a final concentration of 10 μM and incubated at 37°C with rotation for 30 min. Cells were harvested by centrifugation, washed three times, and resuspended in prewarmed PBSg (PBS with 0.2% glucose). Cells (200 μl) were transferred to a black polystyrene 96-well plate (Greiner; 655076) and monitored (ex, 360/em, 450 and 500 nm) on a BioTek Synergy H1 plate reader. Baseline fluorescence was recorded for 5 min prior to addition of compound. Fluorescence was recorded for 25 additional minutes. Laurdan generalized polarization (GP) was calculated: GP = (I₄₆₀ − I₅₀₀)/(I₄₆₀ + I₅₀₀).