*[2-14C] Acetic Acid. Perkin Elmer Cat# NEC085H001MC
From PMID 20484008
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.
from PMID 9081212==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 0.1 uCi/mL 14C Acetic Acid)* PBS at 4C* [2-14C] Acetic Acid. Perkin Elmer Cat# NEC085H001MC
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).
Figure 2==Protocol==The monolavers were incubated # 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 20 min >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 CO2 incubatorline other than adipocytes, followed by increase the addition of 2 ~uCi of [~4C]-hot glucose at a final specific activity of to 0.05 mCi/mmol5 uCi per mL. The uptake and incorporation of radio- label was allowed # Pretreat cells with inhibitors if required.# Change Media to proceed for 20 rain, at 37 ° C, '''Acetate Incorporation Media''' and add 100 nM insulin as required.# Place in the CO~ radioactive tissue culture incubator, and was terminated by rapidly washing .# Save 3 x 5 uL of the monolayers '''Acetate Incorporation Media''' to count total radioactivity.# After 60 min wash cells 3x with ice-cold phosphate- buffered saline (1 mL of PBS). The monolayers were then scraped from each dish into # Resuspend cells in 1.5 ml mL of PBS. and the # Transfer 900 uL of resuspended cells were disrupted in 2.1 mL of PBS and 3 mL of non-aqueous betaflour scintillant and vortex. Set aside to let separate# Do a glass Dounce homogenizerbradford assay on 50 uL of lysed cells for protein normalization. Neutral lipids were extracted from the # Add 200 uL of glycogen solution to cells into chlorofnrm:methanol:acetic acid, as previously described (Radinand vortex. 196o: Tarlow et ah# The next day, 1977). After evap- oration move the lipid portion to dryness under nitrogen, the residue was dissolved in an organic solvent-based scintillation fluid a fresh vial and the radioactivity was quantitated by scin- tillation countingcount. Adapted from Matt Brady's protocol.