Difference between revisions of "Acetate Incorporation into Lipid"

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[[Category:mTORC1]]
 
[[Category:mTORC1]]
  
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From PMID 20484008
 
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Lipid synthesis and extraction. Cellular lipids were extracted essentially by the Folch method (6). Briefly, cells were incubated for 2 h at 37°C in the presence of [14C]acetic acid or [14C]glucose, washed once in cold PBS, and then lysed in 0.5 ml of ethanol. One milliliter of chloroform was added and mixed by vortexing. After addition of 0.5 ml of HCl (0.1 N), lysates were mixed by gentle inversion. Tubes were centrifuged at 500 g for 20 min. The upper phase and interface were discarded, and the organic phase was washed twice with 0.5 ml of water. Extracts were dried under N2, and the pellet was resuspended in the appropriate volume of chloroform-methanol (2:1). Extracts were separated by thin-layer chromatography with diethylether-hexane-glacial acetic acid (35:65:1, vol/vol/vol) as carrier solution on silica gel plates and exposed to film. Phospholipids and triacylglycerol were identified with standards.
 
  
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from PMID 9081212
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==Materials==
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* Cells plated in 12 well dishes
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* Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS)
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* Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and 10 uCi/mL 14C Acetic Acid)
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* PBS at 4C
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* [2-14C] Acetic Acid.  Perkin Elmer Cat# NEC085H001MC
  
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The cells were treated as described in the text and in the legend to Fig. 2, except the final concentration of acetate was 2 mM, at a specific activity of 0.1 mCi/mmol of [2-14C]-acetic acid, and was added to complete cell culture media containing 25 mM glucose. In each experiment, insulin resistant and control cell monolayers were assessed in parallel, and each experiment was performed at least four times. These assay condi- tions described for both glucose and acetate resulted in linear incorporation of radiolabel into cellular material for at least 60 rain. in both the presence and absence of insulin (data not shown). 
 
  
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Figure 2
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==Protocol==
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The monolavers were incubated for 20 min in a CO2 incubator, followed by the addition of 2 ~uCi of [~4C]-glucose at a final specific activity of 0.05 mCi/mmol. The uptake and incorporation of radio- label was allowed to proceed for 20 rain, at 37 ° C, in the CO~ incubator, and was terminated by rapidly washing the monolayers with ice-cold phosphate- buffered saline (PBS). The monolayers were then scraped from each dish into 1.5 ml of PBS. and the cells were disrupted in a glass Dounce homogenizer. Neutral lipids were extracted from the cells into chlorofnrm:methanol:acetic acid, as previously described (Radin. 196o: Tarlow et ah, 1977). After evap- oration to dryness under nitrogen, the residue was dissolved in an organic solvent-based scintillation fluid and the radioactivity was quantitated by scin- tillation counting.
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# Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well.
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# Starve cells in '''Low Glucose Starvation Media''' for >3h.
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# 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.
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# Pretreat cells with inhibitors if required.
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# Add 100 nM insulin as required.
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# Add 50 uL of Hot Acetate Solution to each well.
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# Place in the radioactive tissue culture incubator for 60-120 minutes.
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# Save 3 x 5 uL of the '''Acetate Incorporation Media''' to count total radioactivity.
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# After 60 min  wash cells 3x with 1 mL of PBS.
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# Resuspend cells in 1 mL of PBS.
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# 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
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# Do a bradford assay on 50 uL of lysed cells for protein normalization.
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# Add 200 uL of glycogen solution to cells and vortex.
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# The next day, move the lipid portion to a fresh vial and count.
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Adapted from Matt Brady's protocol.

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

  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 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.
  4. Pretreat cells with inhibitors if required.
  5. Add 100 nM insulin as required.
  6. Add 50 uL of Hot Acetate Solution to each well.
  7. Place in the radioactive tissue culture incubator for 60-120 minutes.
  8. Save 3 x 5 uL of the Acetate Incorporation Media to count total radioactivity.
  9. After 60 min wash cells 3x with 1 mL of PBS.
  10. Resuspend cells in 1 mL of PBS.
  11. 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
  12. Do a bradford assay on 50 uL of lysed cells for protein normalization.
  13. Add 200 uL of glycogen solution to cells and vortex.
  14. The next day, move the lipid portion to a fresh vial and count.

Adapted from Matt Brady's protocol.

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