Cholesteryl sulfate

Cholesterol, ergosterol, dihydroCholesterol, coprostanol, and cholesteryl sulfate were purchased from Sigma (St. Louis, MO), epiCholesterol from Steraloids, cholestenone and lanosterol from Avanti Polar Lipids, cholesteryl N-(2-dimethylaminoethyl)carbamate and cholesteryl N-(trimethyl-ammonioethyl) carbamate chloride from Santa Cruz Biotechnology. Sterol stocks were prepared at 20 mM in chloroform, except for cholesteryl sulfate which was prepared in methanol, stored at −20 °C and pre-warmed to 25 °C prior to use. Methyl-β-cyclodextrin (MβCD)-sterol complexes were formed in phosphate buffered saline (10 mM phosphate/90 mM citrate/150 mM NaCl, pH 7.4; PBS-citrate) at a molar ratio of 1:2. Sterol suspensions were sonicated for 30 s and incubated at 37 °C overnight, at which point any insoluble sterol was removed via 0.2 μm filtration.

FL, lignoceric acid, palmitic acid, boric acid, potassium chloride, sodium hydroxide, chloroform, methanol, and ethanol were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Cholesterol, cholesteryl sulfate, octacosanoic acid, 1,6-diphenyl-1,3,5-hexatriene (DPH), N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenyl-ammonium p-toluenesulfonate (TMA-DPH), and 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) were from Sigma-Aldrich (St. Louis, MO, USA). Ceramide type III and ceramide type VI were from Evonik Industries AG (Essen, Germany). These reagents were used without further purification.