Cholesterol fluorometric assay kit

First, dissolve stearylamine, cholesterol and phenamil proportionally in benzene/methanol (90/10 v/v). Freeze the above solutions in liquid nitrogen, and lyophilize them overnight to completely eliminate the organic solvent.[26 (link)] Then, Hydrate the freeze-dried mixtures in a pH 7.4 TRIS buffer (TRIS 50 mM, NaCl 140 mM). Carry out five cycles from liquid nitrogen temperature to ~70 °C, and vortex between successive cycles to obtain well-hydrated samples. Finally, liposomes were obtained with probe sonicator (20 s on and 5 s off) for 20 min.
A Sephadex G-50 spin column (diameter 0.4 cm, length 7 cm) was centrifuged at 3500 rpm for 1 min to separate free phenamil from phenamil-containing liposomes.[27 ] Phenamil and cholesterol content of the collected fraction were quantified using UV-Vis spectrometer and Cholesterol Fluorometric Assay Kit (Cayman Chemical, Ann Arbor, MI).
siRNA and Sterosomes complexation was prepared and validated according to our previous work.[24 (link)] Briefly, Sterosomes and siRNA were diluted separately in Tris-NaCl buffer in Eppendorf tubes and kept at room temperature for 5 min. The contents of two tubes were combined and incubated at room temperature for 20 min.
A Malvern Zetasizer was employed to determine the hydrodynamic diameters and the zeta potential of obtained Sterosomes at 25 °C.

Cholesterol content in specific lipoprotein particles was analyzed using fast protein liquid chromatography (FPLC) as previously described (Rigotti et al. 1997 (link)) with minor modifications. Pooled plasma (50 μl total from five mice) was subjected to FPLC using Superose 6 Increase 10/300 GL columns (Sigma, CAT# GE29-0915-96) with elution buffer (154 mM NaCl, 0.02% sodium azide). Proteins were eluted at 0.6 ml/min. Forty fractions were collected, eluted and total cholesterol in each fraction was determined using Cholesterol fluorometric assay kit (Cayman Chemical, CAT#10007640).