The rate of DNL was measured by the amount of uptake of 1-[14C]-acetate into the lipid component of hepatocytes, as described previously [25] (link). After serum starvation, hepatocytes were incubated for 12 hours in serum free media containing Exendin-4 (10 nM or 100 nM). 1-[14C]-acetic acid [0.12 μCi/L; Perkin Elmer) with unlabeled sodium acetate [10 μM] was added to the treated serum-free media for an additional 6 hours. After incubation cells were washed and scraped into 250 ml of PBS (1×). The lipid fraction was recovered in Folch solvent (chloroform:methanol 2:1), the solvent was evaporated and the 14C radioactivity retained in the cellular lipid was determined by scintillation counting and expressed as disintegrations per minute (dpm)/per well.
1 14c acetic acid
Manufactured by PerkinElmer
Sourced in United States
[1-14C]acetic acid is a radioactive isotope of acetic acid used as a tracer in various scientific research applications. It provides a means to track the movement and incorporation of the acetic acid molecule in biological and chemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Tri carb liquid scintillation analyzer, by Hewlett-Packard (1 mentions)
Tlc silica gel 60 f254, by Merck Group (1 mentions)
Haemin, by Merck Group (1 mentions)
Monoclonal anti polyhistidine alkaline phosphatase antibody, by Merck Group (1 mentions)
1 14c oleic acid, by PerkinElmer (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cysteine, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
1 14c lauric acid, by American Radiolabeled Chemicals (1 mentions)
Aconitase assay kit, by Cayman Chemical (1 mentions)
6 14c glucose, by PerkinElmer (1 mentions)
Citrate synthase assay kit, by Merck Group (1 mentions)
Succinate dehydrogenase activity colorimetric assay kit, by Abcam (1 mentions)
Isocitrate dehydrogenase activity assay kit, by Merck Group (1 mentions)
Alpha ketoglutarate assay kit, by Abcam (1 mentions)
14c l glutamine, by PerkinElmer (1 mentions)
Dowex 1x2 400 resin, by Merck Group (1 mentions)
A769662, by Bio-Techne (1 mentions)
S 4 nitrobenzyl 6 thioinosine nbmpr, by Bio-Techne (1 mentions)
D u 14c glucose, by PerkinElmer (1 mentions)
9 protocols using 1 14c acetic acid
1
Quantifying Lipid Synthesis in Hepatocytes
2
Measuring Glucose Uptake and Lipogenesis
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Glucose uptake was measured using [1,2-3H]-2-deoxy-d -glucose as previously described [43] (link). De novo lipogenesis was determined by [1-14C]-acetic acid incorporation into triglycerides. The day before the assay, insulin was removed from the culture medium. Cells were incubated for 3 h in Krebs–Ringer buffer (cf. Section 2.1.5 ) supplemented with 2% BSA, 10 mM HEPES, 2 mM glucose, 5 mM acetate and 2 μCi [1-14C]-acetic acid (PerkinElmer), in the presence or absence of 100 nM insulin. Cells were scraped in STED buffer and neutral lipids were separated by thin layer chromatography.
3
Lipid Synthesis in TSC2-Deficient Cells
4
Lipogenesis Quantification via 14C-Acetate Assay
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To check the lipogenesis, a well-established 14C-acetate incorporation assay was used6 (link). SV-sebocytes were treated with bilobetin for 1 day, then 2 µCi of [1-14C]acetic acid (PerkinElmer, Boston, MA, USA) was added and incubated for a further 6 hours. Cellular lipids were extracted with the solvent comprising of chloroform and methanol (2:1). After evaporation of solvent, cellular lipids were dissolved in chloroform and separated using a thin layer chromatography (TLC silica gel 60 F254; Merck KGaA). The developing buffer for TLC consists of hexane and ethyl acetate (6:1). Lipids were visualized by autoradiography.
5
Comprehensive Lipid Biosynthesis Assay
6
Tracing glucose flux through the TCA cycle
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Glucose flux through the TCA cycle was measured by radiolabelling 1 × 106 cells with 2 μCi [6-14C]-glucose (55 mCi/mmol; PerkinElmer, Waltham, MA, USA), [1-14C]-acetic acid (1 mCi/ml, PerkinElmer) or [14C]-L-glutamine (200 mCi/mmol, PerkinElmer). Cell suspensions were incubated for 1 h in a closed experimental system to trap the 14CO2 released from [14C]-glucose, [14C]-acetic acid], and [14C]-L-glutamine. The reaction was stopped by injecting 0.5 ml of 0.8 N HClO4. The amount of glucose transformed into CO2 through the TCA cycle was calculated as described in [103 (link)].
The enzymatic activity of citrate synthase, aconitase, IDH, alpha-ketoglutarate dehydrogenase, and SDH were measured on the basis of 10 µg of mitochondrial proteins using a citrate synthase assay kit (Sigma), an aconitase assay kit (Cayman Chemical, Ann Arbor, MI), an isocitrate dehydrogenase activity assay kit (Sigma), an alpha-ketoglutarate assay kit (Abcam), and a succinate dehydrogenase activity colorimetric assay kit (BioVision), as per the respective manufacturer’s instructions.
The enzymatic activity of citrate synthase, aconitase, IDH, alpha-ketoglutarate dehydrogenase, and SDH were measured on the basis of 10 µg of mitochondrial proteins using a citrate synthase assay kit (Sigma), an aconitase assay kit (Cayman Chemical, Ann Arbor, MI), an isocitrate dehydrogenase activity assay kit (Sigma), an alpha-ketoglutarate assay kit (Abcam), and a succinate dehydrogenase activity colorimetric assay kit (BioVision), as per the respective manufacturer’s instructions.
7
Assays for Cellular Fatty Acid Oxidation and Lipogenesis
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For fatty acid oxidation, primary MEFs were serum starved for 3 h and incubated overnight in culture medium containing 100 mM palmitate (C16:0) and 1 mM carnitine. In the final 2 h of incubation, cells were pulsed with 1.7 µM Ci[9,10(n)-3H] palmitic acid (GE Healthcare), and the medium was collected and eluted on ion exchange columns packed with DOWEX 1X2-400 resin (Sigma) to analyze the released 3H2O, formed during cellular oxidation of [3H] palmitate. Primary MEFs used for this assay were below passage 5.
For the measurement of lipogenesis, A549 cells were cultured, placed overnight in low-glucose low-serum medium, then labeled with 1-14C acetic acid (Perkin Elmer) while stimulated with serum-containing medium for 1 h. Cells were washed twice with PBS before lysis in 0.5% Triton X-100. The lipid fraction was isolated by the addition of chloroform and methanol (2:1 [v/v]), followed by the addition of water. Samples were centrifuged, and 14C incorporation was measured in the bottom, lipid-containing phase using a scintillation counter. Each condition was normalized to protein concentrations.
For the measurement of lipogenesis, A549 cells were cultured, placed overnight in low-glucose low-serum medium, then labeled with 1-14C acetic acid (Perkin Elmer) while stimulated with serum-containing medium for 1 h. Cells were washed twice with PBS before lysis in 0.5% Triton X-100. The lipid fraction was isolated by the addition of chloroform and methanol (2:1 [v/v]), followed by the addition of water. Samples were centrifuged, and 14C incorporation was measured in the bottom, lipid-containing phase using a scintillation counter. Each condition was normalized to protein concentrations.
8
Protocols for Metabolic Assays and Compound Sources
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5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, #OR1170T) was obtained from Apollo Scientific. 991 (#AOB8150) was purchased from Aobious. Insulin (#I9287) and SBI-0206965 (#SML1540) were obtained from MerckMilliporeSigma. A769662 (#3336) and S-(4-Nitrobenzyl)-6-thioinosine (NBMPR, #2924) were purchased from Tocris Bioscience. Deoxy-d -[1, 2-3H]-glucose, [5, 6-3H]-uridine, [1-14C]-Acetic acid and [U-14C]-d -glucose were purchased from PerkinElmer. Rotenone (#R8875) was obtained from MerckMilliporeSigma. Cell culture reagents were purchased from Thermo Fisher Scientific. All other materials, unless otherwise indicated, were from MerckMilliporeSigma.
9
Fungal Biomass and Growth Estimation
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Fungal growth was estimated using the method of Bååth et al. (2001) with adaptations by Rousk et al. (2009) , which make use of the ability of fungi to incorporate acetate into ergosterol. For this, 0.4 g fresh soil or minerals were transferred into a 10 ml glass tube and a mixture of 20 μl 1-[ 14 C]acetic acid (sodium salt, 2.04 GBq mmol -1 , 7.4 MBq ml -1 , Perkin Elmer, USA) and unlabeled acetate was added, resulting in a final acetate concentration of 220 μM. Samples were incubated for 6 h at 20 °C in the dark. After phase separation and specific preparation steps, the quantity of ergosterol was measured using HPLC with a UV detector (282 nm) (LaChrom Elite system with a Chromolith column (VWR-Hitachi)) and methanol (2 ml min -1 ) as the mobile phase. The ergosterol fraction was separated with a fraction collector as described by Rousk and Bååth (2007) . The incorporated 14 C acetate in ergosterol was measured using liquid scintillation. Ergosterol concentration corresponded to the fungal biomass, whereas the detected radioactivity, expressed as picomoles per gram soil per hour, represented fungal growth.
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