Cholesterol biosynthesis assay

From LipidomicsWiki

Cholesterol biosynthesis assay using [3H]acetic acid
(used in Huh7 human hepatoma cells, but should work in any cultured cells)

1. Plate cells on 6-well plates in Eagle-MEM (Sigma) supplemented with 10% fetal bovine serum (FBS), 20mM HEPES, pH 7.4 without antibiotics.
2. If you want to manipulate gene expression in the cells, plasmid transfection can be done for 24 h using Lipofectamine 2000 (Invitrogen) or siRNA transfection for 48 h using HiPerfect (Qiagen) or Interferin (Polyplus) according to the manufacturers’ instructions
3. Wash cells with serum-free medium.
4. Pulse label cells for 30 min at +37oC with [3H]acetic acid (500μCi/well; AmershamBiosciences TRK12) in serum-free medium
5. Wash 3x with serum-free medium
6.. Chase 90 min with 25mM mevalonate (Sigma) in serum-free medium.
7. After washing with PBS, scrape cells into 900 μl of ice-cold 2% (w/v) NaCl.
8. Add [14C]cholesterol (roughly 100 000 DPM) to the cell lysate to be able to correct the results for material loss.
9. Withdraw a 100 μl aliquot for protein analysis (BioRad DC assay).
10. From the remaining 800 μl, extract lipids:
     Bligh and Dyer extraction: Add 2 ml methanol and 1 ml chloroform, vortex.
     Centrifuge 1000xg 10 min.
     Transfer the supernatants to fresh tubes.
     Add 1 ml water and 1 ml chloroform, vortex.
     Centrifuge 1000xg 10 min.
     Transfer the lower phase to a fresh tube
     Evaporate the samples with nitrogen gas.
     Dissolve in 50µl chloroform, rinse tube with another 25 µl chloroform, combine
11. Separate extracted lipids by TLC (Merck silica gel 60 plates, cat. no. 1.05721.0001) by using petroleum ether /diethyl ether/acetic acid (60:40:1) as the solvent
12. Dry plates and stain them with iodine vapor. Identify cholesterol band based on comigration with a cholesterol standard
13. Scrape cholesterol band into scintillation vials, measure [3H]- and [14C] radioactivity by liquid-scintillation counting.
14. Correct results for protein amount and for material losses (using the [14C]radioactivity)