Primary hepatocytes were isolated from the indicated mice (10–15 week old males) by collagenase perfusion and cultured as previously described (43 (link), 61 (link)). Briefly, the cells were plated in M199 medium containing Glutamax and supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 10% (v/v) fetal calf serum, 500 nM dexamethasone (MilliporeSigma), 100 nM triiodothyronine (MilliporeSigma), and 10 nM insulin (MilliporeSigma). The hepatocytes were attached for 4 h and then maintained in M199 medium with antibiotics and 100 nM dexamethasone for 16 h. The experiments were performed the following morning by treating with the compounds (as indicated in the figure legends) followed by cell lysis for Western blot analysis or lipogenesis assay. Rate of de novo lipogenesis in mouse primary hepatocytes was measured as incorporation of [14C] into fatty acids using [1-14C]-acetate (PerkinElmer) as substrate (43 (link), 47 (link)).
1 14c acetate
Manufactured by PerkinElmer
Sourced in United States
[1-14C]-acetate is a radiolabeled compound used in research applications. It contains a carbon-14 isotope incorporated at the first carbon position of the acetate molecule. This product can be used as a tracer to study various biochemical processes involving acetate metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Ls6500, by Beckman Coulter (2 mentions)
Imagequant tl, by Cytiva (2 mentions)
Liquid scintillation counter, by Merck Group (2 mentions)
5735 silica gel 60f254, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Triiodothyronine, by Merck Group (1 mentions)
Liquid scintillation counter, by Beckman Coulter (1 mentions)
Typhoon fla 7000, by GE Healthcare (1 mentions)
Tlc silica gel 60 f254, by Merck Group (1 mentions)
Myristic acid, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions)
1 14c oleate, by PerkinElmer (1 mentions)
High density nickel resin, by GoldBio (1 mentions)
Fatty acid free bsa, by Merck Group (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (1 mentions)
Storm 820 phosphorimager, by Cytiva (1 mentions)
High performance tlc silica gel 60 plates, by Merck Group (1 mentions)
19 protocols using 1 14c acetate
1
Isolation and Culture of Primary Mouse Hepatocytes
2
Analysis of Fatty Acid and Mycolic Acid Biosynthesis
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FA and MA biosynthesis were analysed by incorporation of [1-14C] acetate. Mycobacterium smegmatis mabR cKD and WT-pMT13 were grown with ATc 2.5 and 25 ng ml−1 and incubated at 37°C with gentle shaking (180 r.p.m.). At T1, aliquots of 5 ml were radiolabelled with 1 µCi ml−1 of [1-14C] acetate (50.5 mCi mmol−1; PerkinElmer) for 1 h at 37°C. Cells were then harvested by centrifugation, washed with phosphate buffer 0.1 M (pH 7.6) and stored at −80°C. FA methyl esters (FAMEs) and MA methyl esters (MAMEs) were obtained after treatment of the radiolabelled cell pellets containing the same number of cells with aqueous tetrabutyl ammonium hydroxide followed by esterification with methyl iodide and extraction with dichloromethane, as previously described [32 ]. The resulting solution of FAMEs and MAMEs was assayed for radioactivity in a Beckman liquid scintillation counter and then equal counts were subjected to thin-layer chromatography (TLC), using silica gel plates (TLC silica gel 60 F254, Merck) and hexane : ethyl acetate (9 : 1, v/v) as the developing solvent. The radioactivity of FAMEs and MAMEs on the TLC plates was visualized using laser scanner Typhoon FLA 7000 (GE Healthcare). The spots of 14C-labelled FAMEs and MAMEs were quantified using Gel -Pro Analyzer software and the values obtained were expressed as arbitrary units (AU).
3
Acetate Incorporation in Hepatocytes
4
Fatty Acid Synthesis in Primary Hepatocytes
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Mouse primary hepatocytes in 6-well plates (4 × 105 cells/well) and human primary hepatocytes in 12-well plates (1.5 × 104 cells/well) were maintained in M199 medium containing antibiotics and 100 nM dexamethasone for 16 h before the evaluation of fatty acid synthesis. Hepatocytes were then incubated for 3 h with 0.6 μCi/ml [1-14C]-acetate (55.3 mCi/mmol) (Perkin Elmer) in fresh M199 medium containing various concentrations of AMPK activators or TOFA, as described in the figure legends. The cells were then scrapped into 1 ml of PBS and transferred to tubes containing 1 ml of 40% KOH. An equal volume of methanol was added and the tubes were heated at 80 °C for 1 h. Non-saponifiable and saponifiable lipids were extracted with petroleum ether before and after acidification with HCl, respectively. Lipid fractions were washed with 5% acetic acid and the solvent was allowed to evaporate off to dryness. The samples were dissolved in liquid scintillation fluid for the determination of [14C] incorporation. Data were normalized against protein content and are expressed in micromoles acetate incorporated per gram of protein per hour or as a percentage of condition incubated in the absence of compounds.
5
Radiolabeled Fatty Acid Metabolism Assay
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Fatty acid-free BSA, myristic acid, oleic acid, palmitic acid, and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich (St. Louis, MO). Antibiotics, high-density nickel resin, and IPTG were purchased from GoldBio (St. Louis, MO). [1-14C]acetate (55 mCi/mmol) and [1-14C]oleate (56.3 mCi/mmol) were purchased from PerkinElmer (Waltham, MA). Bacteria media supplies were purchased from BD Medical Technologies (Franklin Lakes, NJ). All chemicals and solvents were reagent grade or better.
6
Isotopic Labeling of Mycobacterial Strains
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The parental and mutant strains were grown at permissive temperature in 7H9-Gly-ADS-Tw broth until an OD600 of 0.6, at which point each culture was divided into two aliquots and 1-[14C] acetate (sodium salt; 58.9 millicurie (mCi)/mmol; Perkin Elmer, Boston, MA) was added (1 μCi mL−1; 37 kiloBecquerel (kBq) mL−1) to each of them, followed by further incubation for 3 h at 30°C or 42°C. The resulting 14C-labeled cells were harvested by centrifugation at 5,000 × g, washed twice with distilled water, and kept frozen until use.
7
Lipid Composition Analysis of E. coli Strains
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The lipid composition of the different E. coli strains was determined by triplicate following labeling with [1-14C]acetate (Perkin Elmer). LB cultures (1.5 mL) were inoculated to an initial optical density at 600 nm (OD600) of 0.1 from precultures grown in the same medium. After the addition of 1 μCi/mL [1-14C]acetate to each culture, they were incubated for 24, 48 or 72 h. The cells were harvested by centrifugation and resuspended in 100 μL of water. The lipids were extracted according to the method of Bligh and Dyer [51 (link)]. The chloroform phase was used for lipid analysis by one-dimensional thin-layer chromatography (TLC) using high-performance TLC silica gel 60 plates (Merck) and ethyl acetate-hexane-acetic acid (60:40:5 [vol/vol/vol]) as the mobile phase. Two-dimensional TLC was performed as described previously [52 (link)]. Radioactivity was detected using a Storm 820 PhosphorImager (Amersham Biosciences). Image analysis and signal quantification were carried out using ImageQuant TL (Amersham Biosciences).
8
Triacylglycerol Biosynthesis in Mycobacterium
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The biosynthesis of triacylglycerols (TAGs) was analysed by incorporation of [1-14C] acetate. Mycobacterium smegmatis fas cKD and WT-pFRA42B were grown to OD600 ∼ 0.2 and each culture was split into two equal fractions. ATc 200 ng ml−1 was added to one of the fractions while the other served as the untreated control. All of the cultures were incubated at 42°C with gentle shaking (180 r.p.m.). At different time points during the growth of the cultures, aliquots of 5 ml were radiolabelled with 1 µCi ml−1 of [1-14C] acetate (50.5 mCi mmol−1; Perkin Elmer) for 1 h at 42°C. Cells were then harvested by centrifugation, washed with phosphate buffer 0.1 M pH 7.6 and stored at −80°C. Total lipids were extracted from radiolabelled cell pellets containing the same number of cells, with methanol/chloroform (2 : 1 v/v) as described by Bligh & Dyer [30 (link)]. After extraction, the lipids were dried and analysed by TLC on silica gel 60 F254 plates (Merck), using hexane/diethylether/acetic acid (75 : 25 : 1, v/v/v) as the developing solvent. Autoradiograms were produced by overnight exposure of the TLC plates to Carestream Kodak BioMax MR films.
9
Measuring Cellular Acetate Metabolism
10
Measuring Lipid Synthesis in Yeast
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Cultures in SC were diluted to OD600 0.1 in SC or SC supplemented with 75 µM of inositol and grown for 24 h at 30°C in the presence of 1 µCi/ml [1-14C]acetate (45–60 mCi/mmol; Perkin Elmer). Lipids were extracted as described in the previous section with of 5 mM HCl, resolved by TLC with chloroform/methanol/acetic acid (65:25:8 vol/vol), scanned on a Typhoon Trio phosphorimager (Amersham Biosciences), and quantified with Quantity One (Bio-Rad).
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