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).