Measurement of membrane fluidity, hydration and cellular cholesterol content
After treatment with atorvastatin, phloretin or 6-ketocholestanol, 150,000 cells were stained with 10 µM 4′-(trimethylammonio)-diphenylhexatriene (TMA-DPH) or 2 µM Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine; both obtained from Sigma-Aldrich) for 30 minutes at room temperature. Anisotropy of TMA-DPH was measured with a Fluorolog-3 spectrofluorimeter with the temperature of the cuvette holder adjusted to 37°C. TMA-DPH was excited at 352 nm and its emission was measured at 430 nm. Fluorescence anisotropy (r ) was measured in the L-format according to the following formula (Kuhry, Fonteneau, Duportail, Maechling & Laustriat, 1983):
where I vv and I vh are the vertical and horizontal components, respectively, of the fluorescence excited by vertically polarized light, and G is an instrument-specific correction factor characterizing the different sensitivity of the detection system for vertically and horizontally polarized light. Laurdan was excited at 350 nm and its emission was detected in the blue range of its emission spectrum between 430-440 nm (I blue) and at the red edge between 495-505 nm (I red) to measure generalized polarization (GP ) according to the following formula (Parasassi, De Stasio, Ravagnan, Rusch & Gratton, 1991):
Total cholesterol content of cellular samples was determined using Cholesterol Quantification Kit (MAK043, Sigma-Aldrich).