Cholesteryl bodipy 542 563 c11

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)}

Immunofluorescence assays were performed as follows^{16 (link)}. Briefly, the indicated cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 2.5% bovine serum albumin, reacted with specific primary antibodies and visualized with Alexa Fluor 488-, or 568-conjugated secondary antibodies as appropriate. BODIPY493/503 or CholEsteryl BODIPY™ 542/563 C₁₁ (Thermo Fisher Scientific Inc.) was applied for tracing LDs. The images were captured using a multi-photon laser scanning microscope (FluoView^TM, Olympus, Tokyo, Japan).
Pearson’s correlation and Mander’s overlap coefficients were used as statistics for quantifying co-localization of target proteins in region of interest with Coloc2 plugin from ImageJ/Fiji software^{49 (link)}, the former by assessing the linear relationship of fluorescence intensities between the two images, and the latter by counting the fraction of pixels with co-occurrence of the two fluorescent images which were primarily insensitive to the signal intensities. The fluorescence intensity profile across the arrow for both green and red channels was analysed using Olympus FV31S-SW software (Olympus, Japan).