4 14c cholesterol

Dulbecco's modified Eagle’s medium, RPMI 1640 medium, penicillin/streptomycin, and Geneticin G-418 were purchased from ThermoFisher Scientific (Waltham, MA, USA). Biowest fetal bovine serum (FBS) was provided by VWR (Dietikon, CH). Poly-D-lysine was purchased from Corning (Bedford, MA, USA). L-[methyl-³H]carnitine hydrochloride ([³H]L-carnitine, specific activity: 81.0 Ci/mmol) and ³⁶Chloride (specific activity: 10 mCi/g) were synthesized by Amersham Life Sciences (Piscataway, NJ, USA); [4-¹⁴C]-cholesterol ([¹⁴C]cholesterol, specific activity: 50.8 mCi/mmol) was purchased from PerkinElmer (Boston, MA, USA). Nonlabeled L-carnitine, methyl-β-cyclodextrin (mβcd), and nonlabeled cholesterol were provided by Sigma-Aldrich (St. Louis, MO, USA). Cholesterol-saturated mβcd (RAMEB) was provided by CycloLab Ltd (Budapest, Hungary).

RAW 264.7 macrophages were grown in DMEM, 10% FBS, 10 mM Hepes (pH 7.3). Two 75 cm² flasks of RAW 264.7 macrophages were loaded with 400 µM oleic acid –BSA complex and labeled with 0.1 µM 4-[¹⁴C]cholesterol (specific activity, 53.0 mCi/mmol, Perkin Elmer) for 18 h in DMEM, 10% FBS, 10 mM Hepes (pH 7.3). Cells were washed twice with PBS and treated with 10 U/ml coase for 2 h 37°C in DPBS+ or mock treated in buffer only. Cells were collected in PBS, and lysed in 1.7 ml of hypotonic lysis buffer (HLB) [20 mM Tris–HCl, pH 7.4; 1 mM EDTA] including a protease inhibitor mixture (chymostatin, leupeptin, antipain, and pepstatin A; 20 µg/ml each) on ice. The lysate was centrifuged at 4°C, 1000×g for 10 min to remove the nuclei. Then, 1.3 ml of the supernatant was mixed with 0.65 ml of 60% sucrose solution to reach a final concentration of 20% sucrose. The samples were transferred to SW50 tubes, and overlaid with 1.7 ml 5% sucrose in HLB and 1.3 ml HLB. The samples were centrifuged for 2 h at 28 000×g, and the LD fraction was recovered from the top of the gradient by using a tube slicer; six additional fractions were collected and the fractions were immediately used for lipid extraction.

Cholesterol feeding experiments were carried out at 20 °C on 10 cm agarose plates (1.75% (w/v) and 52 mM NaCl), inoculated with 800 µL of an overnight culture of OP50 E. coli, supplemented with 0.5 µCi of [4-¹⁴C]-cholesterol (Perkin-Elmer, Inc., Waltham, MA, USA). Synchronized L1 larvae, which had hatched overnight on NGM-plates without food and cholesterol, were placed on the feeding plates and allowed to grow to adulthood at 20 °C. The fed animals were washed two times with an M9 buffer, and groups of 20 animals each were either transferred to normal NGM plates or collected for lysis. The animals on the NGM plates were allowed to lay eggs for 3 days at 15 °C, after which they were removed and collected for lysis. After 2 days at 20 °C, the F1 generation was flushed off the plates, washed two times with an M9 buffer, and subjected to lysis. Lysis was carried out for 1 h at 65 °C in a single-worm lysis buffer, containing 0.5 µg/µL proteinase K. Afterwards, all samples were mixed with 5 mL of scintillation liquid Irgasafe plus (Perkin-Elmer, Inc., Waltham, MA, USA), and radioactivity was measured in a Hidex 600 SL liquid scintillation counter (Hidex Oy, Turku, Finland).