915
edits
Changes
increased from 0.1 to 10 uCi per mL.
* 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 0.1 10 uCi/mL 14C Acetic Acid)
* PBS at 4C
* [2-14C] Acetic Acid. Perkin Elmer Cat# NEC085H001MC
# Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well.
# Starve cells in '''Low Glucose Starvation Media''' for >3h.
# 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 0.5 50 uCi per mL.
# Pretreat cells with inhibitors if required.
# Change Media to '''Acetate Incorporation Media''' and add 100 nM insulin as required.