Membrane dipole potential assay

RN cultures were seeded at 4000 cells/well in a 96 well plate and permitted to settle for 24 h before washing well before labelling with 0.5 μM of the fluorescent probe di-8-ANEPPs (Invitrogen, from 2 mM stock solution in ethanol) for 1.5 h in phenol-red free DMEM (Sigma-Aldrich) (Davis et al., 2015 ). After this time the ratiometric di-8-ANEPS fluorescence intensity at excitation of 420/520 nm and emission of 670 nm using a Safire plate reader for each cell population was recorded before and 10 min after cells were treated with varying concentrations of TPGS for 10 min. The change in fluorescence ratio of di-8-ANEPPs indicates a change in the membrane dipole potential on addition of an agent of interest. The dissociation constant (K_d) of the interaction of TPGS for neuronal cells was determined by fitting the change in di-8-ANEPPs fluorescence ratio to a hyperbolic binding equation as described previously (Davis et al., 2010 (link)).