Cholesteryl ester synthesis assay

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3H-oleic acid incorporation into cholesterol esters

THE LABELING MIX:

1. Make serum-free medium containing 2% defatted BSA and sterile filter

2. Add 5 μCi/ml (or desired amount) of 3H-oleic acid (dissolved in ethanol) into the medium. Vortex immediately.

3. Let the labeling mix stand at +37°C at least 30 min (vortex the mix a few times during incubation).

LABELING OF CELLS:

1. Rinse cells 3x with PBS (cells cultured on 12 well plate)

2. Add the labeling mix (if you want to add LDL, drugs etc.:just mix them with the labeling medium before adding on the cells)

3. Put the cells in the incubator at +37°C; the labeling time depends on your cells and conditions but usually 4-6 h will do.

LIPID EXTRACTION:

KEEP ON ICE UNTIL THE SOLVENTS HAVE BEEN ADDED!

1. Take the cells on ice, rinse 4x with cold PBS

2. Scrape the cells with cold 2% NaCl (see step 4) and transfer to a kimax tube (in order to get all the cells collected, do the scraping in 2 steps).

3. Vortex vigorously > let stand for 10 min on ice

4. Vortex and remove aliquots for protein determination, and then adjust the volume to 800 μl
Note: the volumes depend on the amount of cells and the cell type you’re using. Calculate the amount you need for protein determination and then approximate the volumes (for instance: put cells initially into 900 μl of 2% NaCl and remove 50 + 50 μl for Lowry. Or use initially smaller volume, for instance if using fibroblasts, then add NaCl to adjust the volume.)

5. Add 14C-cholesteryl oleate (internal standard) to ~10 000-20 000 dpms per sample.

6. Add 2 ml MeOH and 1 ml CHCl3 and vortex

7. Take 1/10 volume of each sample with a Hamilton syringe into a scintillation tube to assess initial 3H and 14C radioactivity in the sample (count with dual label protocol)

8. Add 1 ml H2O and 1 ml CHCl3 and extract by vigorously vortexing, let stand and vortex again. Centrifuge at 2500 rpm for 10 min
9. Take the lower phase into a KK-tube (add a drop of methanol if it gets cloudy)

10. Evaporate the solvent with N2

11. Dissolve the lipid films into ~30 μl of CHCl3 : MeOH (9:1)

12. Apply samples to TLC along with cholesterol ester standard (~300 nmol). Run with hexane/diethyl ether/acetic acid 80 : 20 : 1 as solvent

13. Stain the standard with iodine vapour, scrape the cholesteryl ester bands into scintillation tubes.

14. Count with dual label program, correct for procedural losses according to 14C-standard and plot against the amount of protein in the sample.

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