Membrane Fluidity of Brain Endothelial Cells

Brain endothelial cells from wild type and ApoB-100 transgenic mice were treated overnight with 10 µg/ml LDL or 10 µg/ml oxLDL. Control cells received culture medium. After treatment, cells were collected by trypsinization, washed once with PBS, resuspended in Ringer-Hepes buffer and counted. The density of the cells for the membrane fluidity tests was optimized by absorbance measurement to OD₃₆₀ = 0.05 (Hewlett Packard 8452A Diode Array Spectrophotometer). Cells were labeled with 0.2 μM TMA-DPH (1-(4 trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; Life Technologies, USA). Fluorescence anisotropy was measured on a T-format fluorescence spectrometer (Quanta Master QM-1, Photon Technology International, USA). Excitation and emission wavelengths were 360 and 430 nm (6 nm slits). Cells were kept under a continuous stirring at 37°C [25 (link), 26 (link)]. Anisotropy data were acquired in every second for 10 min. After measuring baseline anisotropy, we introduced a strong membrane fluidizer, benzyl alcohol (30 mM, Merck, Germany) as a positive control. Transgenic and wild type data were calculated and plotted as treatment vs. fluorescent anisotropy.