Difference between revisions of "Acetate Incorporation into Lipid"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (updated with detailed protocol) |
Davebridges (Talk | contribs) (rewrote procedure) |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
[[Category:Lipid Synthesis]] | [[Category:Lipid Synthesis]] | ||
[[Category:mTORC1]] | [[Category:mTORC1]] | ||
| − | |||
| − | |||
| − | |||
| Line 10: | Line 7: | ||
* 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 | ||
| Line 18: | Line 15: | ||
# 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 | + | # Prepare '''Acetate Incorporation Media''' Make at least enough for one extra well, using 50 uL/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. | ||
| − | # | + | # Add 100 nM insulin as required. |
| − | # Place in the radioactive tissue culture incubator. | + | # Add 50 uL of Hot Acetate Solution to each well. |
| + | # Place in the radioactive tissue culture incubator for 60-120 minutes. | ||
# Save 3 x 5 uL of the '''Acetate Incorporation Media''' to count total radioactivity. | # Save 3 x 5 uL of the '''Acetate Incorporation Media''' to count total radioactivity. | ||
# After 60 min wash cells 3x with 1 mL of PBS. | # After 60 min wash cells 3x with 1 mL of PBS. | ||
Latest revision as of 18:45, 10 August 2011
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
- 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 50 uL/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.
- Add 100 nM insulin as required.
- Add 50 uL of Hot Acetate Solution to each well.
- Place in the radioactive tissue culture incubator for 60-120 minutes.
- Save 3 x 5 uL of the Acetate Incorporation Media to count total radioactivity.
- After 60 min wash cells 3x with 1 mL of PBS.
- Resuspend cells in 1 mL of PBS.
- 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
- Do a bradford assay on 50 uL of lysed cells for protein normalization.
- Add 200 uL of glycogen solution to cells and vortex.
- The next day, move the lipid portion to a fresh vial and count.
Adapted from Matt Brady's protocol.
