Sackler School of
Graduate Biomedical Sciences

Sterol Synthesis

Day 0 Seed cells in 12-well plates in medium with full serum. About 20,000 cells/well.
Day 1 Refeed cells medium with LPDS.
Day 3 Refeed cells medium with LPDS. Add 10-30 uCi [³H]acetate. Aftre 2 hr, bring the plates into the cold room and wash the cells 1x, 1 x 10 min, 1x with 3 ml TBS. Aspirate well.

Extract lipids with 1.5 ml hexane:isopropanol (3:2) for 15 min on rotator in cold room. Bring plates into lab and add recovery standard (2000 dpm [¹⁴C]cholesterol, 20 ug cholesterol in 10 ul of chloroform). Put on rotator at RT for addition 15 min.

Transfer extracts into 13x100 mm glass test tubes. Rinse wells with 1 ml hex:IP and pool extracts. Dry lipids on N-Evap.

To measure total sterol synthesis, you must saponify the cholesteryl esters that have formed during the pulse. Add 1 ml of 1 M KOH in ethanol to the extracted cell lipids. Vortex. Heat for 1 hr at 80^oC (heat block on LOW #10 with marbles on top). Cool, add 1 ml dw.

Extract with 2 ml of petroleum ether (or heptane). Transfer PE phase (top) to 13x100 tube. Repeat PE extraction. Backwash PE by adding 1 ml dw and vortexing. Remove lower water layer with long Pasteur pipet and discard. Dry PE on N-Evap.

TLC with freshly made heptane:ethyl ether (90:60) using half TLC plates. Cut out iodine sensitive spot corresponding to cholesterol.

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

You should get 50-100 dpm/ug protein.

References: Liscum, Ruggiero and Faust (1989); Liscum et al. (2002)
Modified September 2006