Tma dph

Culture samples (1 ml) were collected and pelleted by centrifugation at room temperature at 6,010 x g in a tabletop microfuge. The supernatants were removed using aspiration and resuspended in ~10 μl PBS containing the indicated dyes at the following final concentrations: 2.0 μg/ml DAPI DNA stain (Molecular Probes); 0.02 mM TMA-DPH (Life Technologies) or 3.0 μg/ml FM4-64 (Life Technologies). After resuspension, samples were mounted on glass slides with polylysine-treated coverslips. Exposure times were generally 1 sec. Images were collected with a Nikon Ti-E microscope equipped with a CFI Plan Apo lambda DM 100X objective, and Prior Scientific Lumen 200 Illumination system, C-FL UV-2E/C DAPI, and C-FL Texas Red HC HISN Zero Shift filter cubes, and a CoolSNAP HQ2 monochrome camera. All obtained images were captured with NIS Elements Advanced Research (version 4.10), and processed with ImageJ64 [39 ].

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.

POPE and 1,2-diacyl-sn-glycero-3-phosphoethanolamine from soybean (SoyPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. TMA-DPH was purchased from Molecular Probes, Inc. (Eugene, OR, USA). HEPES buffer contains 10 mM HEPES, 150 mM NaCl, 1 mM EDTA (pH 7.4). LUVs were extruded with Whatman® nuclepore polycarbonate filters (Sigma-Aldrich, St. Louis, USA) in a mini-extruder (Avanti Polar Lipids, Alabaster, USA). Turbidity was measured in a spectrophotometer (Beckam Instruments, Inc., Fullerton, USA), whereas steady-state fluorescence anisotropy measurements were made using a Horiba Jobin Yvon Spex Fluorolog 3-22/Tau 3 spectrofluorometer (Kyoto, Japan). MLVs containing 0.4 mol% TMA-DPH were obtained by co-dissolving the lipids (POPE:SoyPE 3:1), probe, and glycoside in chloroform, followed by solvent evaporation under nitrogen stream and high vacuum desiccation. Dried lipid films were then hydrated with 10 mM HEPES buffer (pH 7.4) to the desired final concentration (0.5 mM lipid, and 0/50 μM glycoside). Lipid suspensions were subjected to five cycles of freezing and thawing at 25 °C. Anisotropy was measured by exciting samples at 360 nm and collecting emission at 430 nm, using 2.7 nm slit width. Temperature was maintained with an isothermal bath and was monitored with a thermistor, inserted into the sample chamber.