Laurdan Membrane Fluidity Assay

The membrane fluidity of S. mutans was measured using laurdan (AnaSpec, Fremont, CA, United States) essentially as described (Bessa et al., 2019). Laurdan is a fluorescence probe that intercalates into the membrane bilayer and displays an emission wavelength shift depending on the amount of water molecules in the membrane (Wenzel et al., 2018). S. mutans (OD₆₀₀ = 0.3 nm) was treated with different concentrations of CBG (0, 4, 6, 8, 10, and 20 μg/ml) at 37°C for 2 h and then incubated with 10 μM laurdan for 10 min at room temperature in the dark. An unstained sample served as control. Thereafter, the samples were washed four times in PBS containing 1% glucose and 1% DMSO (PBSGD) and resuspended in 1 ml of PBSGD. 200 μl of each sample were added to each well of a μClear black 96-well plate (Greiner Bio-One, Frickenhausen, Germany) and the fluorescence analyzed at 30°C in the M200 Tecan plate reader with an excitation at 350 nm and an emission scan from 400 to 600 nm. The laurdan Generalized Polarization (GP) was calculated using the following equation: GP = (I₄₄₀−I₄₉₀)/(I₄₄₀ + I₄₉₀) where I₄₄₀ and I₄₉₀ are fluorescence intensities at 440 and 490 nm, respectively.