1,6-Diphenyl-1,3,5-hexatriene

Method for Using the 1,6-Diphenyl-1,3,5-hexatriene Fluorescent Probe in Membrane Studies^[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Dissolve 1,6-Diphenyl-1,3,5-hexatriene in dimethylformamide (DMF) to prepare a stock solution; dissolve POPC (34.6mM) and ITZ (9.5mM) separately in chloroform to prepare stock solutions.
2. Liposome Preparation (Small Unilamellar Vesicles, SUVs): Mix appropriate volumes of POPC and ITZ chloroform stock solutions in a volumetric flask, evaporate the solvent under a stream of nitrogen to form a dry lipid film. Hydrate with phosphate buffer (final POPC concentration of 2.5mg/mL), and sonicate to obtain SUVs with a particle size of 30-200nm.
3. 1,6-Diphenyl-1,3,5-hexatriene Labeling: Add the DPH stock solution to the liposome sample to achieve a final 1,6-Diphenyl-1,3,5-hexatriene concentration of 102nM, and incubate in the dark for 1 hour.
4. Steady-State Fluorescence Anisotropy Measurement: Use a spectrofluorometer, set the excitation wavelength λ_exc=360nm and emission wavelength λ_em=430nm, and measure the fluorescence intensities of vertical and horizontal polarizations.
Precautions:
(1) The fluorescence lifetime of 1,6-Diphenyl-1,3,5-hexatriene is sensitive to the polarity (dielectric constant) of the membrane environment; the lifetime value increases in more ordered and hydrophobic membrane environments.
(2) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:
[1] Poojari C, Wilkosz N, Lira RB, et al. Behavior of the DPH fluorescence probe in membranes perturbed by drugs. Chem Phys Lipids. 2019 Sep;223:104784.