Laurdan

hN2 cells were grown at 37 C in 5% CO₂ in AB2 basal neural medium supplemented with 2% ANS neural medium, 1% L- glutamine and penstreptomycin and 10μg/μl Leukemia inhibitory factor (LIF). Cells were plated on a 35 mm Fischer Scientific glass-bottom petri dishes coated with Matrigel. NIH3T3, HEK293 and L6 cells were grown at 37C in 5% CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1% Pen-Strep, and 2.5 mL of 1 M HEPES. Freshly split cells were plated onto 35-mm Fischer scientific glass-bottom dishes coated with Matrigel. The membrane dye Laurdan (6-dodecanoyl-2-dimethylamino naphthalene; Invitrogen) was dissolved in dimethylsulfoxide (DMSO), and 2.5 mM stock solution was prepared and added to the cell dishes at final concentration of 10μM. Cells were grown devoid of Laurdan and incubated with the dye prior to imaging. Since growth media can have an effect on GP measurements and produce artifacts, due to the presence of serum that contains lipids which can interfere with the probe’s fluorescence, we used serum free media. To avoid autofluorescence and further ensuing corrections, we used a high enough Laurdan concentration of 10μM [32 (link)].

The membrane probe Laurdan (6-dodecanoyl-2-dimethylamino naphthalene, D250, Invitrogen) was dissolved in DMSO to create a stock solution of 9.19 mM. HEK293T cells were incubated with 1.8 µM Laurdan for 1 hr at 37°C. Generalized polarization (GP) is a ratiometric method which is used to quantitatively report membrane order in living cells. The GP value is calculated as follows: ${\displaystyle Generalizedpolarization=\frac{I_{blue}-I_{green}}{I_{blue}+I_{green}}}$ where I_blue and I_green are the fluorescence intensities emitted at 440 nm and 490 nm, respectively. Conventionally, 440 nm and 490 nm are the emission maximums for ordered lipid and disordered lipid bilayers, respectively. Images were acquired with a LSM780 (Zeiss) laser scanning microscope using a 63X oil immersion objective, coupled to a two-photon Ti:Sapphire laser (Coherent) tuned to 780 nm and 80 MHz. An SP 760 nm dichroic filter was used to separate laser light from fluorescence signal. The fluorescence signal was acquired from 416 nm to 474 nm for the blue channel and from 475 nm to 532 nm for the green channel using the GaAsP PMT detectors of the LSM780. GP values were calculated in Fiji (ImageJ) as previously described (Rentero et al., 2019 (link)). GP values were calculated for each cell of interest and the numeric difference in GP values for the whole cell are presented normalized to empty vector (set to 0).

The stock suspension of 100 μM LUV₁₀₀ was labeled with 0.2 μM concentration of 2-dimethylamino-6-lauroyl naphthalene (Laurdan, Molecular Probes, dissolved in dimethylsulfoxide, DMSO) at 37 °C for 60 min, the final ratio of Laurdan per lipid was 1 : 500. Final concentration of DMSO was 0.2% in all samples. The LUV suspension was split to individual aliquots of 0.1 mL. To study the possible shift in packing of the lipids in membranes with two different compositions (DOPE : DOPG and DOPG : DOPE, 2 : 1, w/w), we measured the generalized polarization (GP) of Laurdan-labeled LUVs under stable temperature 27 °C using FluoroMax-3 spectrofluorometer (Jobin Yvon, Horiba). Fluorescence spectra were measured from 400 to 600 nm (after excitation at 365 nm), with 4 nm bandpass. GP values were calculated according to Parasassi et al. (1990).^{28 (link)}

Formic acid, methanol, AA (L(+) ascorbic acid), AAPH (2,2′-azobis(2-amidinopropane)hydrochloride), MC540 (merocyanine 540), and enzyme COX-2 (cyclo-oxygenase) were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile was purchased from Merck (Darmstadt, Germany). Quercetin-3-O-glucoside, quercetin 3-O-galactoside, quercetin-3-O-rutinoside, (+)catechin, (−)epicatechin, procyanidin B2-3-O-gallate, procyanidin B3, vitexin, and luteolin-3-O-glucoside were purchased from Extrasynthese (Lyon, France). The DPH-PA (3-(4-(6-phenyl)-1,3,5-hexatrienyl) phenylpropionic acid), Laurdan ((6-dodecanoyl-2-dimethylaminonaphthalene)), and DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence probes were purchased from Life Technologies (California, USA).

Dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol were purchased from Sigma-Aldrich. Bovine brain sphingomyelin (SM) was from Avanti Polar Lipids (Alabaster, USA). Laurdan was purchased from Life Technologies. Dye PA was synthesized as described elsewhere35 (link).