Topfluor pc

All lipids (dioleoyl phosphatidylcholine, DOPC: dioleoyl phosphatidylserine, DOPS; TopFluorPE; RhPE, TopFluorPC, and TopFluo TMR PC) were purchased from Avanti Polar Lipids. Large unilamellar vesicles (LUVs) were prepared by extrusion. In brief, lipids dissolved in benzene/methanol (95:5, vol:vol) were freeze-dried under high vacuum overnight and the dried lipid powder was hydrated at room temperature in a buffer containing 100 mM NaCl, 10 mM Hepes, 5 mM EDTA, pH 7.0 and vortexed. The resulting lipid suspension was submitted to 10 successive cycles of freezing and thawing by successively immersing the vial containing the lipid suspension into liquid nitrogen and a warm water bath. Thereafter the lipid suspension was extruded 10 times through two stacked polycarbonate filters of 100 nm pore-size (Nucleopore, Whatman) using a LIPEX extruder (Northern Lipids Inc., Burnaby, Canada) to produce large unilamellar vesicles.

LUVs were prepared at a total
lipid concentration of 1.0 mg/mL, as described above. TRO, SQ, and
ENT-03, when present, were added during the hydration phase to a final
concentration of 5 μM. BODIPY-FL C5-ganglioside GM1 (GM1-D),
BODIPY-FL-cholesterol (CHOL-D), BODIPY-FL-sphingomyelin (SM-D, commercial
name TopFluor Sphingomyelin, Avanti Polar Lipids), and BODIPY-FL-DOPC
(DOPC-D, commercial name TopFluor PC, Avanti Polar Lipids) were used
as donor lipids. Cholesteryl 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate
(CHOL-A, commercial name CholEsteryl BODIPY 542/563 C11, ThermoFisher
Scientific) was used as an acceptor lipid. The molar fraction of each
lipid labeled with D or with A was 0.0625% of total lipids in all
cases.
Fluorescence spectra of LUVs containing only lipid-D,
only CHOL-A, and both lipid-D and CHOL-A were acquired using the cell
and spectrofluorometer described above, at 25 °C, with excitation
at 450 nm and emission from 480 to 640 nm. FRET efficiencies (E) were calculated as where F_DA is the fluorescence intensity of D in the presence of A,
and F_D is the fluorescence intensity of
D in the absence of A.⁴⁴ FRET E calculated with eq 12 was converted into distance between D and A (r)
using:⁴⁴ where R₀ is the Forster distance and was previously calculated for
this D/A probe pair.^{11 (link)}

Phosphate Buffered Saline (PBS) was prepared from tablets (Sigma-Aldrich, Denmark). One tablet dissolved in 200 mL of MilliQ water yielded 0.01 M phosphate buffer with 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C.
All other chemicals used were from Sigma-Aldrich (Denmark). The fluorescent dyes used in confocal microscopy were: 1-palmitoyl-2-(dipyrrometheneborondifiuoride)undecanoyl-sn- glycero-3 phosphoethanolamine, TopFluor PC (Avanti Polar Lipids, USA), Rhodamine B (Sigma-Aldrich, Denmark), Fluorescein isothiocyanate, FITC (Thermo Fischer Scientific, USA) and Rhodamine B DHPE (Sigma-Aldrich, Denmark). The embedding medium used in confocal microscopy and GSD-Raman was Pro-Long Diamond Slowfade reagent (Life Technologies, Denmark).