Reagents:

• ¹⁴C Acetic acid sodium salt, Amersham CFA.13, 0.2 µCi/µl

• 1x PBS

• H₂O

• Chloroform, Methanol, Hexane, Isopropanol, 4 L, Stores

• Long glass tubes and glass pipettes

• Micropipette, VWR 53432-783

• TLC Silica gel 60 (20 x 20 cm), VWR EM-11845-7, 25/$231.53

• Carrier lipid stocks; UC (unstearified cholesterol) 1 mg/ml, CE (cholesterol ester) 0.5 mg/ml, TG (triglyceride) 1 mg/ml.

• Neutrolipid solvent (N.L.); 108 ml hexethane, 1.2 ml HAc (glacial), 30 ml Diethyl Ether (anhydrous); available at Stores

• Iodine Crystals

• Scintillation Fluid (Ecolume) and vials (Wheaton #986644)

Protocol:

1. Set up cultures at 5 x 10⁵ cells/ml in 2 ml with ¹⁴C acetate (10 µl at 0.2 µCi/µl – final 1 µCi/µl).

2. Grow 1 to 2 hours.

3. Wash with 1x PBS (important to remove FBS).

4. Store cell pellet at -20°C until ready for further processing.

5. Add 1 ml Hexane:Isopropanol 3:1 v/v (to extract lipids from pellet).

6. Vortex.

7. Incubate 30 minutes at RT.

8. Transfer supernatant to new glass tube careful not to touch the pellet.

9. Wash pellet with 1 ml Hexane:Isopropanol. Transfer supernatant to same glass tube.

10. Evaporate to dryness under air stream in 40°C heat block.

11. Add 4 ml Folch (chloroform:methanol 2:1 v/v).

12. Add 1 ml H₂O.

13. Add 20 µg complete carrier lipid to each sample.

14. Vortex.

15. Incubate 20-30 minutes at RT.

16. Vortex.

17. Spin for 10 minutes at 2000 rpm.

18. Aspirate top aqueous phase, keep organic phase.

19. Add 1 ml H₂O.

20. Vortex.

21. Spin for 10 minutes at 2000 rpm.

22. Aspirate top aqueous phase, keep organic phase.

23. Evaporate to dryness under air stream in 40°C heat block.

24. Rinse sides with chloroform.

25. Evaporate to dryness under air stream in 40°C heat block.

26. Resuspend in 110 µl chloroform.

27. Spot at ~1.5 cm from bottom of gel (above tank solvent).

    1. Spot carrier alone for a reference band.

1. Place gel in tank with solvent and run.

   1. Run until gel is completely saturated ~20 min.

   2. Dry gel on 40°C heat block.

1. Place gel in tank of fresh iodine crystals until all bands are clearly visible.

2. Mark bands of interest with pencil.

3. Dry gel until iodine stained bands are clearly not visible.

4. Cut marked bands into scintillation vials and add 4.5 ml scintillation fluid.

Reference: Oram Lab, Medicine, Metabolism Endocrinology, University of Washington

Reagents:

• ¹⁴C mevalonic acid DBED salt, NEC166, 0.1 µCi/µl

• 1x PBS

• H₂O

• Glass tubes, 16 ml, VWR 60828-387, 72/$134.02

• Glass tubes, 50 ml, VWR 60828-423, 36/$112.25

• Chloroform, 4 L, Stores

• Methanol, 4 L, Stores

• N₂ gas stream

• Petroleum ether (35-60°C), 1 L, Stores 0014-285, $8.99

• Na₂SO₄ (anhydrous), 3 kg, Stores 0004-410, $28.09

• Glass pasteur pipette

• Glass wool

• Toluene (purified), 4 L, Stores 0015-070, $17.45

• Ethyl ether (anhydrous), 1 L, Stores 0012-625, $17.14

• TLC silica gel 60 (20 x 20 cm), VWR EM-11845-7, 25/$231.53

• Iodine (crystal), VWR MK098402, 125 g/$98.40

Protocol:

1. Set up culture at 10⁶ cells/ml in 100ml with ¹⁴C mevalonate
   (50 µl at 0.1 µCi/µl – final 0.05 µCi/ml).

2. Grow 0 to 48 hours.

3. Wash with 1x PBS (important to remove FBS).

4. Resuspend in 1ml H₂O. Use glass tubes from here on.

5. Add to 10ml chloroform:methanol (2:1 v/v).

6. Stir at RT for 3 hours.

7. Evaporate under N₂ gas stream for ~1 hour until solution turns cloudy.

8. Extract 2x with 10 ml petroleum ether at 40-60°C.

9. Lower fraction ~500 µl, transfer upper fraction (x2) to 50 ml tube.

10. Dry with anhydrous Na₂SO₄.

11. Filter through pasteur pipette with glass wool.

12. Evaporate to dryness under N₂.

13. Store at -20°C until use.

14. Place 100 ml solvent in tank (toluene:diethylether 9:1 v/v).

15. Resuspend samples in 100 µl petroleum ether (vaporizes rapidly).

16. Spot at ~1.5 cm above bottom of gel.

17. Place gel in tank and run about 1.5 hrs or until solvent front reaches about one inch from the top of the plate.

18. Remove from tank and dry overnight to prevent ¹⁴C contamination of phosphor-screen.

19. Cover in plastic wrap and expose on a phosphor-screen to visualize ¹⁴C-labeled compounds.

20. To visualize markers, sprinkle crystals of iodine on the bottom of a sealable glass container.

21. Place plates in container and seal lid.

22. Wait 20-30 minutes for entire plate to be exposed.

23. Circle visualized spots as staining will quickly fade.

24. Calculate R_f value by dividing the distance traveled of substance by the distance traveled of solvent front.