Acetate Incorporation into Lipid

Materials

• Cells plated in 12 well dishes
• Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS)
• Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and 10 uCi/mL 14C Acetic Acid)
• PBS at 4C
• [2-14C] Acetic Acid. Perkin Elmer Cat# NEC085H001MC

Protocol

1. Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well.
2. Starve cells in Low Glucose Starvation Media for >3h.
3. Prepare Acetate Incorporation Media Make at least enough for one extra well, using 0.5 mL/well for a 12 well dish. If monitoring lipogenesis in a line other than adipocytes, increase the hot glucose to 50 uCi per mL.
4. Pretreat cells with inhibitors if required.
5. Change Media to Acetate Incorporation Media and add 100 nM insulin as required.
6. Place in the radioactive tissue culture incubator.
7. Save 3 x 5 uL of the Acetate Incorporation Media to count total radioactivity.
8. After 60 min wash cells 3x with 1 mL of PBS.
9. Resuspend cells in 1 mL of PBS.
10. Transfer 900 uL of resuspended cells 2.1 mL of PBS and 3 mL of non-aqueous betaflour scintillant and vortex. Set aside to let separate
11. Do a bradford assay on 50 uL of lysed cells for protein normalization.
12. Add 200 uL of glycogen solution to cells and vortex.
13. The next day, move the lipid portion to a fresh vial and count.

Adapted from Matt Brady's protocol.