2.4. Steady-state fluorescence anisotropy

Another measure of membrane order used here is the steady-state fluorescence anisotropy of diphenylhexatriene (DPH). DPH preferentially localizes in the hydrophobic region of the membrane. As such, the orientation and rotational freedom of the DPH molecules in this region reports on the order of the lipid fatty acyl chains. This can be quantified by calculating the anisotropy, 〈r〉, of DPH:

where I is the fluorescence intensity at different excitation and emission polarizer combinations, denoted by the subscripts, respectively, where V is vertical and H is horizontal, and G is a correction factor to account for detector sensitivity.

Anisotropy measurements were recorded with an Agilent Cary Eclipse fluorometer. After cells were treated with MLT, they were resuspended in PBS and incubated with 10 μM DPH for ∼45 minutes at 37 °C. Samples were excited at 350 nm and emission was collected at 430 nm. Measurements were taken at room temperature.