Sackler School of
Graduate Biomedical Sciences

Transport of plasma membrane [³H]cholesterol to the endoplasmic reticulum as measured by [³H]cholesterol esterification

Day 0 Seed cells in 12-well plates at 15,000 cells/well in H-NCS.
Day 2 Refeed H-NCS or H-NCLPDS as needed for experiment.
Day 3 Refeed all wells 0.5 ml/well H-NCS or H-NCLPDS.
Make staggered additions of 0.5 or 1 uCi [³H]cholesterol to triplicate wells.

For example

Harvest at 6 pm. 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) for 15 min on cold room rotator. Bring plates to lab, add 10 ul recovery standard. Incubate for 15 min on lab rotator. Transfer organic extracts to 12X75 glass tubes. Wash wells with 1 ml hexane/isopropanol (3:2). Pool extracts and dry using N-evap.

Spot samples on 20x20 cm plastic-backed silica gel G plates that have been cut in half. TLC with toluene/ethyl acetate (2:1). Visualize samples in iodine tank. Cholesteryl ester Rf = 0.75. Cholesterol Rf = 0.25. 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: Underwood et al. (1998)
Modified September 2006