Inositol Labeling and Lipid Extraction

�*Cells in 100 mm culture dishes �*Labelling Media (Inositol Free DMEM with PSG, 10% dialysed serum and 100 uCi inositol per plate of cells) �*Perchloric acid (640 uL into 10 mL water) keep cold �*100 mM EDTA (2 mL of 0.5M stock in 10 mL) keep cold �*Water �*Deacylation Mix (3.5 mL Methylamine, 6 mL Methanol, 1.5 mL Butanol, 2.1 mL Water) �*Lipid Extraction Mix (10 mL Butanol, 2 mL Ethyl Ether and 0.5 mL Ethyl Formate)

1. Label subconfluent cells (60-80% confluence) for 48h in labelling media. If using adipocytes label at 4-6 days post-FBS for 48h
2. Check that eppendorf centrifuge is at 4C and that heating block (with right size holes) is at 56C
3. Treat cells as required(remove media if necessary)
4. Aspirate media and add 1 mL Perchloric acid. Incubate at 4 C for 15 min
5. Scrape cells, transfer to eppendorf tubes and spin 10 min at 16 000g at 4 C
6. Aspirate supernantant and resuspend cells in 1 mL 100 mM EDTA. Centrifuge again.
7. Suspend pellet in 50 uL of water.
8. Deacylation
   1. Add 1 mL Deacylation mix and transfer to glass tube
   2. Incubate at 56 C for 45 min
   3. Dry under vacuum
9. Resuspend pellet in 0.5 mL water. Vortex 30s
10. Centrifuge at room temperature for 2 min
11. Extract Lipids:
    1. Remove supernatant to tube with 0.5 mL Lipid Extraction Mix
    2. Vortex 15s
    3. Centrifuge at room temperature for 2 min
    4. Remove lower phase and repeat steps a-c once
12. Remove lower phase to clean tube and dry under vacuum.
13. Store dried samples at -20 C

[1] C C Whiteford, C Best, A Kazlauskas, and E T Ulug. D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor. Biochem J, 319 ( Pt 3):851{60, Nov 1996.