Acetate Incorporation into Lipid: Difference between revisions
m removed duplicate listing |
increased from 0.1 to 10 uCi per mL. |
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* Cells plated in 12 well dishes | * Cells plated in 12 well dishes | ||
* Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS) | * Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS) | ||
* Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and | * Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and 10 uCi/mL 14C Acetic Acid) | ||
* PBS at 4C | * PBS at 4C | ||
* [2-14C] Acetic Acid. Perkin Elmer Cat# NEC085H001MC | * [2-14C] Acetic Acid. Perkin Elmer Cat# NEC085H001MC | ||
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# Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well. | # 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. | # 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 | # 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. | ||
# Pretreat cells with inhibitors if required. | # Pretreat cells with inhibitors if required. | ||
# Change Media to '''Acetate Incorporation Media''' and add 100 nM insulin as required. | # Change Media to '''Acetate Incorporation Media''' and add 100 nM insulin as required. | ||