Membrane dipole potential measurement with di-8-ANEPPS
After treatment with atorvastatin, 6-ketocholestanol or phloretin cells were incubated with di-8-ANEPPS (Thermo Fisher, D3167) at a final concentration of 2 µM on ice for 20 minutes (Clarke & Kane, 1997; Gross, Bedlack & Loew, 1994; Kovács, Batta, Zákány, Szöllősi & Nagy, 2017). Cells were kept on ice until measurement. Right before spectrofluorimetry, the sample was warmed to 37°C, and spectra were acquired with a Fluorolog-3 spectrofluorimeter (Horiba Jobin Yvon, Edison, NJ). The temperature of the cuvette holder was adjusted to 37°C by a circulating water bath. Excitation spectra were registered between 410 nm and 520, while the emission wavelength was set to 660 nm in order to minimize effects of membrane fluidity on the fluorescence of the indicator (Clarke & Kane, 1997). The ratio of fluorescence intensities integrated between excitation wavelengths 410-416 nm and 503-510 nm correlates positively with the dipole potential (Clarke & Kane, 1997; Kovács, Batta, Zákány, Szöllősi & Nagy, 2017).