Di 4 aneppdhq

The dyes Di-8-ANEPPS
(Biotium, catalog no. 61012), F2N12S (3-hydroxyflavone, ThermoFisher,
catalog no. A35137) and Di-4-ANEPPDHQ (ThermoFisher, catalog no. D36802)
were prepared as recommended by the vendor. Dye solutions were then
filtered with a 0.2 μm regenerated cellulose syringe filter
(Corning, catalog no. 431215) and individual aliquots were stored
at −20°C. Working solutions were prepared in DMSO (Millipore
Sigma, catalog no. D8418) to a concentration of 5 mM. Only the aliquot
actively being used was stored short-term at 4 °C. This 5 mM
stock was diluted fresh in DMSO before each experiment to make the
25 μM working stocks used for staining buffers. Unless stated
otherwise, the final staining concentration was 2 μM.

In order to perform lipid packing imaging, immature and mature moDCs (day 7) were spiked with a final concentration of 0.4 μM with Di-4-ANEPPDHQ (ThermoFisher) and subsequently incubated for 5 min on ice (77 (link)). In the next step, cells were washed with PBS. RPMI1640 without phenol red and serum was applied to the cells and the solution was transferred into 8-well chamber slides for imaging. The spectral imaging was performed using a Zeiss LSM780 confocal microscope equipped with a 40×, 1.2NA objective, and a 32-channel GaAsP detector array. Fluorescence excitation of Di-4-ANEPPDHQ was set to 488 nm and the lambda detection range was set between 500 and 700 nm. The values of the 32 channels were analyzed within the ImageJ plug-in “Stacks-T functions-Intensity vs. Time Monitor”. To calculate generalized polarization (GP) values, one has to define the wavelengths λ_Ld and λ_Lo of maximum emission of a probe in a reference liquid‐disordered (Ld) and liquid‐ordered (Lo) membrane environment. The wavelengths λ = 650 nm as maximum wavelength in disordered membraned (red shifted) and λ = 560 nm as maximum wavelength in the gel phase (blue shifted) were used as described previously (77 (link)–79 (link)).

The HepG2 cell line was purchased from the American Type Culture Collection. Fetal bovine serum was purchased by EUROCLONE (ECS0180DH), while L-glutamine (25030081) and penicillin-streptomycin (15140122) were purchased by GIBCO. DMEM Medium (D6046), Sodium chloride (NaCl) (S9888), Calcium chloride (CaCl₂) (C1016), HEPES (H3375), Hydrochloric acid (HCl) (1.01514), Sodium hydroxide (NaOH) (S8045), Paraformaldehyde (PFA) (16005), Dithiothreitol (DTT) (D9779), and Nile Red (19123) were purchased from Sigma-Aldrich (Sofia, Bulgaria). Transportan 10 (TP10) and TP10 labeled with carboxyfluorescein (CF) at the N-terminus (CF-TP10) were purchased from the CRIBI-Peptide Facility, University of Padova. di-4-ANEPPDHQ (D36802) was purchased from Thermo Fisher Scientific (Waltham, MA USA).

Rat hippocampal neurons were cultured 14 and 28 days in vitro (DIV) and fixed in 4% PFA. For lipid phase imaging, cells were stained with di‐4‐ANEPPDHQ (Thermo Fisher) according to the manufacturer's specifications. For imaging, we used a Nikon A1R confocal microscope attached to Ti eclipse outfitted with a Plan Apo VC 60× lens, oil immersion lens, with a NA of 1.4. For imaging, we used the spectral detector of the system set to 530–590 nm and 590–650 nm to image the spectral shift of the dye from lipid ordered to disordered phase. The resulting images were analyzed according to Owen Rentero Magenau Abu‐Siniyeh and Gaus (2012). To compare the two groups, the average value of 108 (60 for 14 DIV [12/coverslip, 5 experiments] and 48 for 28 DIV [12/coverslip, 4 experiments]) fields of view was reported.

For imaging membrane fluidity, cells were grown on glass coverslips for 48 hours. Cells were stained with di-4-ANEPPDHQ (Thermo Fisher Scientific) following the protocol provided by the manufacturer. Briefly, 5 mM of di-4-ANNEPPDHQ stock solution in DMSO was diluted in serum-free DMEM (final concentration: 5 μM). Cells were incubated with di-4-ANNEPPDHQ-containing DMEM for 30 minutes at 37°C in the dark. Then, cells were washed with DMEM and imaged using an FV3000 confocal microscope with excitation at 488 nm and emission at both 530-590 (ordered lipid) and 590-650 nm (disordered lipid). The images were analyzed using ImageJ to generate ratiometric results of ordered-to-disordered lipids.

The lipophilic voltage-sensitive dye Di-4-ANEPPDHQ (Thermo Fisher Scientific) was bath-applied to stain neuronal membranes [47 (link)]. Stock solutions (5 mM) in dimethyl sulfoxide were aliquoted for single use and kept at −20°C. Immediately before application, solutions were diluted 1:1 with pluronic acid F-127 (20% solution; Biotium, Hayward, CA, US) dimethyl sulfoxide solution and mixed with saline to a final concentration of 50 μM. A petroleum jelly well was built around the desheathed CoG (S1 Fig), and the dye was applied for 30–60 minutes, after which the petroleum jelly well was removed, and the preparation was continuously superfused with cooled (10–13°C) saline for the remainder of the experiment.