Cholesterol cholesteryl ester quantitation assay kit

10 × 10⁶ cells were lysed in 0.5 mL of 10 mM Tris, 100 mM NaCl, 20 mM KH₂PO₄, 30 mM EDTA, 1 mM EGTA, 250 mM sucrose, pH 7.5 and sonicated with 2 bursts of 10 s (Labsonic sonicator, Sartorius Stedim Biotech S.A., Aubagne Cedex, France), then centrifuged at 13000 g for 15 min at 4 °C. The supernatants were centrifuged at 100000 g for 1 h at 4 °C, using an Optima L-90K Beckman Coulter Ultracentrifuge (Beckman Coulter Inc, Fullerton, CA) to collect the membrane fractions. The pellets were resuspended in 250 µL of the assay buffer provided by fluorimetric Cholesterol/Cholesteryl Ester Assay Kit – Quantitation (Abcam) and used to measure free cholesterol in the membrane, as per manufacturer’s instructions. An aliquot of 50 µl was sonicated again to measure the membrane proteins. Results were expressed as mg cholesterol/mg membrane proteins.

Sk-Hep1 cells, BTECs, or LSECs were lysed in 0.5 mL of lysis buffer (HEPES, pH 7.4, 20 mM, KCl 10 mM, MgCl₂ 2 mM, EGTA 1, and DTT 1 mM) by gentle scraping. Following an incubation time of 15 min on ice, the lysate was passed through a 25-gauge needle 10 times and kept on ice for a further 20 min. To remove nuclei and mitochondria, samples were centrifuged at 10,000× g for 5 min at 4 °C. The supernatant was subsequently ultra-centrifuged (Beckman Coulter’s, rotor 70Ti) at 100,000× g for 1 h at 4 °C. The new supernatant (i.e., the cytoplasm fraction) was discarded, whereas the pellet (i.e., the membrane fraction) was rinsed with 200 µL of PBS and kept at −80 °C until further processing. The protein content of the membrane extracts was measured on a 50 µL aliquot, using the BCA Protein Assay kit (Sigma Aldrich). The amount of cholesterol was measured on 100 µg membrane protein using the fluorimetric Cholesterol/Cholesteryl Ester Assay Kit—Quantitation (Abcam, Cambridge, UK), as per the manufacturer’s instructions. The results are expressed as µmol cholesterol/mg membrane protein, based on the calibration curve. Membrane cholesterol assay was performed in three independent experiments.

The amount of cholesterol was measured in 100 µg total protein by using the fluorimetric Cholesterol/Cholesteryl Ester Assay Kit–Quantitation (Abcam), according to the manufacturer’s instructions. The results are expressed as µmoles cholesterol/mg cell protein, according to the calibration curve.