Sackler School of
Graduate Biomedical Sciences

Cholesterol esterification

Day 0 Seed cells in multiwell plates in medium with full serum.
Day 1 Refeed medium with LPDS.
Day 2-3 Make additions of LDL and/or oxysterols.

Pulse cells with [³H]oleate (10 ul per ml of media) for 1-2 hr. Final concentration of oleate should be 0.1 mM; approximately 30,000 dpm/nmol.

At end of pulse, bring plates into the cold room. Aspirate media and wash dishes with TBS once quickly, once for 10 min on rotator, and once quickly with 3 ml each. Aspirate TBS completely. Extract lipids with 2 ml hexane/isopropanol (3:2) on cold room rotator for 15 min. Bring plates to the lab, add 10 ul recovery standard and incubate for 15 min on the lab rotator.

Transfer the organic extracts to 12X75 glass tubes. Wash wells with additional 1 ml hexane/isopropanol (3:2). Pool extracts and dry using N-evap. Spot on 20x20 cm plastic-backed silica gel G plates that have been cut in half. TLC with heptane/diethyl ether/glacial acetic acid (90:30:1). Visualize samples in iodine tank. Cholesteryl oleate Rf = 0.75. Triolein Rf = 0.55. Cut out spots, put in scintillation vial and add 5 ml ReadySafe. Let vials sit for 30 min and give them a shake before starting to count.

To measure protein: Let plates air dry; add 1 ml 0.1 N NaOH and swirl 1 hour at RT. Transfer 100-200 ul aliquots (depends on cell confluence) to 12x75 tubes. Set up BSA standard tubes with 0, 5, 10, 20 ul 1 mg/ml BSA and 200 ul of 0.1N NaOH. Add 1 ml Lowry reagent, vortex, inc 10 min. Add 50 ul Folin:phenol, vortex, inc 30 min. Read OD 670 nm.

Reference: Munn et al. (2003)
Modified September 2006