Cholesterol fluorimetric assay kit

Tumors were homogenized in 750 µL PBS using the Tissue Lyser II device (Qiagen), as per manufacturer’s instructions, then sonicated (two bursts of 10 s; Labsonic sonicator, Sartorius Stedim Biotech S.A., Aubagne Cedex, France). 500 µL were centrifuged at 13000 g for 15 min at 4 °C. The supernatants were centrifuged at 100000 g for 1 h at 4 °C, using a Optima L-90K Beckman Coulter Ultracentrifuge (Beckman Coulter Inc, Fullerton, CA) in lysis buffer (10 mM Tris, 100 mM NaCl, 20 mM KH₂PO₄, 30 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7.5) supplemented with protease inhibitor cocktail set III, 1 mM Na₃VO₄, 1 mM NaF, 1 mM4-(2-aminoethyl)benzenesulfonyl fluoride (PMSF), 10 mM aprotinin and 10 mM dithiothreitol (DTT). The pellet (corresponding to microsomal fraction) was re-suspended in 500 µl lysis buffer and stored at −80 °C until use. 250 µl was used for the measurement of HMGCR activity, and 250 µL of tumor homogenates before the ultracentrifugation was used to measure total cholesterol. For both total and membrane-associated cholesterol, we used the Cholesterol Fluorimetric Assay kit (Cayman Chemical, Ann Arbor, MI) as per the manufacturer’s instructions. Results were expressed as µmol cholesterol/mg cell proteins.

To prepare cholesterol/methyl-β-cyclodextrin (MβCD) complexes, cholesterol was dissolved in 2-propanol/chloroform 2:1 (v/v) at a final concentration of 6.5 mM and added to an aqueous solution of 66.7 mM MβCD previously heated at 80 °C for 20 min. During the overnight evaporation of the solvent, cholesterol is progressively associated with MβCD in a soluble mixture. In cholesterol loading assays, 5 × 10⁶ cells (after overnight starvation) were incubated with 0.67 mM MβCD for 4 h, then washed; fresh medium containing 0.5 mM cholesterol/MβCD complexes was added for 24 h. In cholesterol depletion assays, cells were incubated with 0.67 mM MβCD for 4 h. After these incubation times, the cellular cholesterol was measured using the Cholesterol Fluorimetric Assay kit (Cayman Chemical, Ann Arbor, MI) as per the manufacturer’s instructions. Results were expressed as µmol cholesterol/mg cell proteins.